Histochemical and morphological evaluation of a glyoxal acid-free fixative.

The application of most chemical fixatives, such as formalin, in the anatomic pathology laboratory requires safety training and hazardous chemical monitoring due to the toxicity and health risks associated with their use. Consequently, the use of formalin has been banned in most applications in Europe; the primary exception is its use in the histology laboratory in lieu of a suitable and safer alternative. Glyoxal based solutions, several of which are available commercially, are the most promising alternative fixatives, because they are based on a mechanism of fixation similar to that of formalin. Unlike formalin, however, glyoxal based solutions do not dissociate from water and therefore do not require ventilation measures such as a fume hood. A primary barrier to the adoption of commercially available glyoxal based solutions is their low pH, which can produce undesirable morphological and antigenic tissue alterations; however, a recently available neutral pH glyoxal product (glyoxal acid free) (GAF) has been developed to mitigate the challenges of low pH. We compared the morphology and histochemistry among tissues fixed in 10% neutral buffered formalin, a commercially available acidic glyoxal product (Prefer), and GAF. Tissues fixed in formalin and Prefer exhibited similar morphology and staining properties; tissues fixed with 2% GAF exhibited deleterious effects.

Glyoxal fixation: An approach to solve immunohistochemical problem in neuroscience research.

The gold-standard fixative for immunohistochemistry is 4% formaldehyde; however, it limits antibody access to target molecules that are buried within specialized neuronal components, such as ionotropic receptors at the postsynapse and voltage-gated ion channels at the axon initial segment, often requiring additional antigen-exposing techniques to detect their authentic signals. To solve this problem, we used glyoxal, a two-carbon atom di-aldehyde. We found that glyoxal fixation greatly improved antibody penetration and immunoreactivity, uncovering signals for buried molecules by conventional immunohistochemical procedures at light and electron microscopic levels. It also enhanced immunosignals of most other molecules, which are known to be detectable in formaldehyde-fixed sections. Furthermore, we unearthed several specific primary antibodies that were once judged to be unusable in formaldehyde-fixed tissues, allowing us to successfully localize so far controversial synaptic adhesion molecule Neuroligin 1. Thus, glyoxal is a highly effective fixative for immunostaining, and a side-by-side comparison of glyoxal and formaldehyde fixation is recommended for routine immunostaining in neuroscience research.

Saline dry fixation for improved cell composition analysis using Raman spectroscopy.

Raman spectroscopy enables the label-free assessment of cellular composition. While live cell analysis is the most accurate approach for cellular Raman spectroscopy, the analysis of fixed cells has proved to be very useful, particularly in collaborative projects where samples need to be serially examined by different laboratories or stored and reanalyzed at a later date. However, many chemicals that are widely used for cell fixation directly affect cellular biomolecules, yielding Raman spectra with missing or altered information. In this article, we compared the suitability of dry-fixation with saline versus chemical fixatives. We compared the Raman spectroscopy of saline dry-fixed cells with the more commonly used formaldehyde and methanol fixation and found that dry-fixed cell spectra preserved more cellular information than either chemical fixative. We also assessed the stability of dry-fixed cells over time and found that they were stable for at least 5 months. Finally, a comparison of dry-fixed and live cell spectra revealed effects due to the hydration state of the cells since they were recovered upon rehydrating dry-fixed samples. Thus, for fixed cell Raman spectroscopy, we recommend dry-fixation with unbuffered saline as a superior method to formaldehyde or methanol fixation.

Natural fixatives alternative to formalin in histopathology: A systematic review.

INTRODUCTION: Since constant long-term exposure to formaldehyde endangers the health of laboratory personnel, sugar-based natural products have become interesting alternative fixatives to formaldehyde because of their preservative and antibacterial properties. However, there are controversial findings on the fixative effects of natural fixatives. This study systematically reviews the evidence comparing natural fixatives' types, dilutions, fixative properties and staining quality in normal tissues and histopathological specimens. MATERIALS AND METHODS: A comprehensive search was performed for studies comparing the natural fixatives- and formaldehyde-fixed tissues using databases from inception to January 2022: PubMed, Ovid Medline and Google Scholar. Two independent reviewers did data extraction. The data were pooled for the type of natural fixatives, their concentrations and fixative qualities compared to formaldehyde. RESULTS: Fifteen studies were included in this systematic review. Nine studies used one natural fixative with different dilutions, while six used several natural fixatives to compare their fixative properties with formaldehyde. The most used natural fixative was honey (n = 12) followed by jaggery (n = 8), sugar (n = 3) and others (n = 1). Honey showed the most promising results in fixation and staining, which are compatible with formalin. Jaggery and sugar also showed the possibility of replacing formaldehyde in tissue fixation and staining in smaller tissue samples. CONCLUSION: Natural fixatives showed promising results in tissue fixation. However, optimising the concentrations and conditions of natural fixatives is difficult because of the different chemical constituents and production steps. More comprehensive studies are necessary for application.

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immunostaining.

Detection of nucleic acids within subcellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics, which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization, and immunostaining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels that escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.

Different fixatives influence morphology, antigen preservation, and TUNEL staining in chicken (Gallus gallus) testis.

The optimized fixative for testis is still controversial. This study investigated the effects of Modified Davidson's Fluid (mDF), 4% Paraformaldehyde (4% PFA), and Bouin's Fluid (BF) fixatives on chicken testes in normal/cadmium (Cd) feeding groups using hematoxylin and eosin (HE), immunohistochemistry (IHC), and Terminal Transferase dUTP Nick End Labeling (TUNEL) staining. Compared to the mDF, we established that the testes fixed with 4% PFA and BF in the normal group had severe shrinkage in tubular and interstitial compartments. Moreover, compared with 4% PFA, the number of GATA4-positive Sertoli cells/mm2 reduced by 67.61% in mDF and 80.57% in BF for one seminiferous tubule. The TUNEL assay illustrated that more positive cells/mm2 in mDF group (28.47 ± 11.38) than in 4% PFA (10.49 ± 7.89). In Cd-treated testes, mDF showed more morphological details than 4% PFA and BF. In contrast, the number of GATA4-positive Sertoli cells/mm2 of 4% PFA was higher than that of mDF by 65.78% and BF by 64.80% in a seminiferous tubule. The number of TUNEL positive cells/mm2 in mDF (272.60 ± 34.41) were higher than in 4% PFA (175.91 ± 19.87). These results suggest that mDF fixative is suitable for normal and Cd-treated testis fixation for HE and TUNEL staining in chicken, whereas 4% PFA fixative is better for IHC examination.

Efficacy of the Antigenicity-Retaining Ability of Fixative Solutions for Liquid-Based Cytology: Immunocytochemistry of Long-Term Storage.

INTRODUCTION/OBJECTIVE: Liquid-based cytology (LBC) is advantageous as multiple stained specimens can be prepared and used for additional assays such as immunocytochemical and molecular-pathological investigations. Two types of preservative-fixative solutions (fixatives) are used for nongynecologic specimens used in the BD SurePath-LBC (SP-LBC) method, and their components vary. However, few studies have evaluated the differences in antigen-retaining ability between these fixatives. Therefore, we investigated and compared the antigen-retaining ability of the fixatives in immunocytochemical staining (ICC) under long-term storage conditions. MATERIALS AND METHODS: Sediments of cultured RAJI cells (derived from Burkitt's lymphoma) were added to each fixative (red and blue) and stored at room temperature for a specified period (1 h; 1 week; and 1, 3, and 6 months). The specimens were then prepared using the SP-LBC method and subjected to ICC. Positivity rate was calculated using the specimens fixed at room temperature for 1 h as a control. Antibodies against Ki67 expressed in the nucleus and against CD20 and leukocyte common antigen (LCA) expressed on the cell membrane were used. RESULTS: For CD20 and LCA, the positivity rate increased with time in the red fixative compared with that in the control. In the blue fixative, the positivity rate was highest at 1 h and was maintained at a high level throughout the storage period. In contrast, the Ki67 positivity rate was highest at 1 h in both red and blue fixatives and markedly decreased with time. Therefore, although refrigerated (8°C) storage was used, no improvement was noted. CONCLUSIONS: Long-term storage is possible for cell membrane antigens at room temperature; however, it is unsuitable for intranuclear antigens. Therefore, we conclude that suitable fixative type and storage temperature differ based on antigen location. Further investigation is warranted.

Evaluation of autofluorescence quenching techniques on formalin- fixed chicken tissues.

Autofluorescence (AF) in formalin-fixed and paraffin-embedded tissues limit their use in immunofluorescence staining techniques. Various methods have been used to reduce AF in human and animal tissues but no protocol has been optimized for avian tissues. The present study was undertaken to evaluate different treatment methods including ammonium chloride, glycine, Trypan blue, sodium borohydride, Sudan Black B, potassium permanganate, LED light, cupric sulphate combined with glycine, ammonium chloride and cupric sulphate in reducing AF in FFPE chicken tissues for the detection of FITC labelled antibodies against immune cell markers. Chicken tissues including conjunctiva, trachea and Harderian gland presented intense non-homogenous AF in cells resembling erythrocytes, connective cells and melanocytes. Only Sudan Black B effectively reduced AF in FFPE tissues; however, no specific fluorescent signal was observed for six FITC labelled antibodies against immune cell markers. Specific fluorescent signal from the FITC-labelled antibodies was observed in frozen chicken tissue sections with minimal AF, suggesting that the AF in FFPE tissues is related to the use of formaldehyde fixatives. In conclusion, this study demonstrates for the first time that AF quenching methods commonly used for other animal species are not appropriate for use in avian tissues and that frozen tissue sections are recommended for immunofluorescence staining techniques in poultry.

RBC-derived vesicles as a systemic delivery system of doxorubicin for lysosomal-mitochondrial axis-improved cancer therapy.

Introduction: Chemotherapeutic drugs are the main intervention for cancer management, but many drawbacks impede their clinical applications. Nanoparticles as drug delivery systems (DDSs) offer much promise to solve these limitations. Objectives: A novel nanocarrier composed of red blood cell (RBC)-derived vesicles (RDVs) surface-linked with doxorubicin (Dox) using glutaraldehyde (glu) to form Dox-gluRDVs was investigated for improved cancer therapy. Methods: We investigated the in vivo antineoplastic performance of Dox-gluRDVs through intravenous (i.v.) administration in the mouse model bearing subcutaneous (s.c.) B16F10 tumor and examined the in vitro antitumor mechanism and efficacy in a panel of cancer cell lines. Results: Dox-gluRDVs can exert superior anticancer activity than free Dox in vitro and in vivo. Distinct from free Dox that is mainly located in the nucleus, but instead Dox-gluRDVs release and efficiently deliver the majority of their conjugated Dox into lysosomes. In vitro mechanism study reveals the critical role of lysosomal Dox accumulation-mediated mitochondrial ROS overproduction followed by the mitochondrial membrane potential loss and the activation of apoptotic signaling for superior anticancer activity of Dox-gluRDVs. Conclusion: This work demonstrates the great potential of RDVs to serve a biological DDS of Dox for systemic administration to improve conventional cancer chemotherapeutics.

Do Tissues Fixed in a Non-crosslinking Fixative Require a Dedicated Formalin-free Processor?

We evaluate the consequences of processing alcohol-fixed tissue in a processor previously used for formalin-fixed tissue. Biospecimens fixed in PAXgene Tissue Fixative were cut into three pieces then processed in a flushed tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) blocks (neutral buffered formalin [NBF]+ve), a formalin-free system (NBF-ve), or left unprocessed. Histomorphology and immunohistochemistry were compared using hematoxylin/eosin staining and antibodies for MLH-1, Ki-67, and CK-7. Nucleic acid was extracted using the PAXgene Tissue RNA/DNA kits and an FFPE RNA extraction kit. RNA integrity was assessed using RNA integrity number (RIN), reverse transcription polymerase chain reaction (RT-PCR) (four amplicons), and quantitative RT-PCR (three genes). For DNA, multiplex PCR, quantitative PCR, DNA integrity number, and gel electrophoresis were used. Compared with NBF-ve, RNA from NBF+ve blocks had 88% lower yield and poorer purity; average RIN reduced from 5.0 to 3.8, amplicon length was 408 base pairs shorter, and Cq numbers were 1.9-2.4 higher. Using the FFPE extraction kit rescued yield and purity, but RIN further declined by 1.1 units. Differences between NBF+ve and NBF-ve in respect of DNA, histomorphology, and immunohistochemistry were either non-existent or small in magnitude. Formalin contamination of a tissue processor and its reagents therefore critically reduce RNA yield and integrity. We discuss the available options users can adopt to ameliorate this problem.

Optimization of Multiple Glycosidase and Chemical Stabilization Strategies for N-Glycan Isomer Detection by Mass Spectrometry Imaging in Formalin-Fixed, Paraffin-Embedded Tissues.

The analysis of N-glycan distributions in formalin-fixed, paraffin-embedded (FFPE) tissues by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is an effective approach for characterization of many disease states. As the workflow has matured and new technology emerged, approaches are needed to more efficiently characterize the isomeric structures of these N-glycans to expand on the specificity of their localization within tissue. Sialic acid chemical derivatization can be used to determine the isomeric linkage (α2,3 or α2,6) of sialic acids attached to N-glycans, while endoglycosidase F3 (Endo F3) can be enzymatically applied to preferentially release α1,6-linked core fucosylated glycans, further describing the linkage of fucose on N-glycans. Here we describe workflows where N-glycans are chemically derivatized to reveal sialic acid isomeric linkages, combined with a dual-enzymatic approach of endoglycosidase F3 and PNGase F to further elucidate fucosylation isomers on the same tissue section.

Using a safe and effective fixative to improve the immunofluorescence staining of bacteria.

The emerging and development of green chemistry has once again drawn the researchers' attention to eliminating the use and generation of hazardous materials. Here we report the use of a safe and effective fixative, chlorine dioxide (ClO2), instead of traditional hazardous fixatives for the cross-linking of cellular proteins to improve immunofluorescence staining of bacteria. The concentration of ClO2needed for 100% fixation is 50µg ml-1, which is much lower than that of traditional fixatives (1000-10000µg ml-1). The ClO2mediated cross-linking can preserve the integrity of bacterial cells and prevent cell loss through lysis. Meanwhile, lysozyme can permeabilize the bacterial cells, allowing the labelled antibodies to diffuse to their intracellular target molecules. By usingE. coliO157:H7/RP4 as a gram-negative bacteria model, immunofluorescence staining assays for both intracellular protein and surface polysaccharide were carried out to investigate the effect of ClO2fixation on the staining. The results demonstrated that ClO2fixation could prevent the target antigens from cracking off the bacteria without damage on the interaction between the antibodies and antigens (either for polysaccharide or protein). As a safe and effective fixative, ClO2has potential practical applications in immunofluorescence staining and fluorescencein situhybridization for single bacteria/cell analysis.

An optimized fixation method containing glyoxal and paraformaldehyde for imaging nuclear bodies.

The mammalian cell nucleus contains different types of membrane-less nuclear bodies (NBs) consisting of proteins and RNAs. Microscopic imaging has been widely applied to study the organization and structure of NBs. However, current fixation methods are not optimized for such imaging: When a fixation method is chosen to maximize the quality of the RNA fluorescence in situ hybridization (FISH), it often limits the labeling efficiency of proteins or affects the ultrastructure of NBs. Here, we report that addition of glyoxal (GO) into the classical paraformaldehyde (PFA) fixation step not only improves FISH signals for RNAs in NBs via augmented permeability of the fixed nucleus and enhanced accessibility of probes, but also largely preserves protein fluorescent signals during fixation and immunostaining. We also show that GO/PFA fixation enables the covisualization of different types of nuclear bodies with minimal impact on their ultrastructures under super-resolution microscopy.

Proteomic comparison between different tissue preservation methods for identification of promising biomarkers of urothelial bladder cancer.

Samples in biobanks are generally preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature compound (OCT)-embedding and subsequently frozen. Mass spectrometry (MS)-based analysis of these samples is now available via developed protocols, however, the differences in results with respect to preservation methods needs further investigation. Here we use bladder urothelial carcinoma tissue of two different tumor stages (Ta/T1-non-muscle invasive bladder cancer (NMIBC), and T2/T3-muscle invasive bladder cancer (MIBC)) which, upon sampling, were divided and preserved by FFPE and OCT. Samples were parallel processed from the two methods and proteins were analyzed with label-free quantitative MS. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Multivariate analysis indicates that the preservation method is the main source of variation, but also tumors of different stages could be differentiated. Proteins involved in mitochondrial function were overrepresented in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Concordant results for proteins such as HMGCS2 (uniquely quantified in Ta/T1 tumors), and LGALS1, ANXA5 and plastin (upregulated in T2/T3 tumors) were observed in both FFPE and OCT data, which supports the use of MS technology for biobank samples and encourages the further evaluation of these proteins as biomarkers.

Robustness of a Cancer Profiling Test Using Formalin-fixed Paraffin Embedded Tumor Specimens.

BACKGROUND/AIM: Cancer profiling tests using formalin-fixed paraffin-embedded (FFPE) specimens with various conditions have become an essential tool for cancer treatment. The robustness of these tests needs to be addressed. MATERIALS AND METHODS: A cancer profiling test, NCC oncopanel, was tested with FFPE specimens from various tissues with different storage conditions and fixation lengths. Next generation sequencing was performed with Miseq and the data were assembled using the human reference genome hg19. RESULTS: Duration of storage and fixation affected the mapping statistics. Prolonged storage increased outward read paring and longer fixation rates caused increased singletons and unmapped reads. CONCLUSION: Our results indicate that a cancer profiling test with target capturing method, NCC oncopanel, shows robustness for FFPE cancer specimens with various storage conditions.

Preservation of histology by phenol-based fixative: mini review of recent findings / Preservación de la histología mediante un fijador basado en fenol: una mini revisión de los hallazgos recientes

SUMMARY: In surgical and anatomical training, use of cadaver remains the most ideal technique. Standard formaldehyde solution preserves cadaveric tissues for an extended period comparing to the unfixed tissues. However, it fails to retain the natural texture, color, and biomechanical features. Phenol based soft embalming methods were developed to maintain these properties, while simultaneously decreasing the biohazard risk. Soft embalming techniques have made the bodies more 'lifelike' and wellfitted for training. Though phenol fixation displays rewarding morphological maintenance, we have scanty evidences on the histological preservation. This mini review primarily discussed the latest reports regarding the effect of phenol-based fixation on the tissue histology. Published literatures revealed phenol-based fixation displayed comparable histological preservation to that ofgold standard paraformaldehyde-based solution. It was concluded that phenol-based solution is an excellent fixative used to preserve tissues for microscopic analysis.

RESUMEN: En el entrenamiento quirúrgico y anatómico, el uso de cadáveres sigue siendo la técnica más ideal. La solución estándar de formaldehído conserva los tejidos cadavéricos durante un período prolongado en comparación con los tejidos no fijados. Sin embargo, no conserva la textura, el color y las características biomecánicas naturales. Se desarrollaron métodos de embalsamamiento blando a base de fenol para mantener estas propiedades y, al mismo tiempo, disminuir el riesgo biológico. Las técnicas de embalsamamiento suaves han hecho que los cuerpos sean más "realistas" y estén mejor preparados para la enseñanza. A pesar que la fijación de fenol muestra un buen mantenimiento morfológico, existe evidencia escasa sobre la preservación histológica. Esta mini revisión se refirió principalmente a los últimos informes sobre el efecto de la fijación en base de fenol en la histología del tejido. La literatura publicada reveló que la fijación a base de fenol mostró una preservación histológica comparable a la de la solución a base de paraformaldehído. Se concluyó que la solución a base de fenol es un excelente fijador utilizado para preservar tejidos para análisis microscópico.

Quantitative Proteomic Analysis Using Formalin-Fixed, Paraffin-Embedded (FFPE) Human Cardiac Tissue.

Clinical tissue archives represent an invaluable source of biological information. Formalin-fixed, paraffin-embedded (FFPE) tissue can be used for retrospective investigation of biomarkers of diseases and prognosis.Recently, the number of studies using proteome profiling of samples from clinical archives has markedly increased. However, the application of conventional quantitative proteomics technologies remains a challenge mainly due to the harsh fixation process resulting in protein cross-linking and protein degradation. In the present chapter, we demonstrate a protocol for label-free proteomic analysis of FFPE tissue prepared from human cardiac autopsies. The data presented here highlight the applicability and suitability of FFPE heart tissue for understanding the molecular mechanism of cardiac injury using a proteomics approach.

Chromosomal Junction Detection from Whole-Genome Sequencing on Formalin-Fixed, Paraffin-Embedded Tumors.

DNA junctions (DNAJs) frequently impact clinically relevant genes in tumors and are important for diagnostic and therapeutic purposes. Although routinely screened through fluorescence in situ hybridization assays, such testing only allows the interrogation of single-gene regions or known fusion partners. Comprehensive assessment of DNAJs present across the entire genome can only be determined from whole-genome sequencing. Structural variance analysis from whole-genome paired-end sequencing data is, however, frequently restricted to copy number changes without DNAJ detection. Through optimized whole-genome sequencing and specialized bioinformatics algorithms, complete structural variance analysis is reported, including DNAJs, from formalin-fixed DNA. Selective library assembly from larger fragments (>500 bp) and economical sequencing depths (300 to 400 million reads) provide representative genomic coverage profiles and increased allelic coverage to levels compatible with DNAJ calling (40× to 60×). Although consistently fragmented, more recently formalin-fixed, specimens (<2 years' storage) revealed consistent populations of larger DNA fragments. Optimized bioinformatics efficiently detected >90% of DNAJs in two prostate tumors (approximately 60% tumor) previously analyzed by mate-pair sequencing on fresh frozen tissue, with evidence of at least one spanning-read in 99% of DNAJs. Rigorous masking with data from unrelated formalin-fixed tissue progressively eliminated many false-positive DNAJs, without loss of true positives, resulting in low numbers of false-positive passing current filters. This methodology enables more comprehensive clinical genomics testing on formalin-fixed clinical specimens.

Innovative Tumor Tissue Dissection Tool for Molecular Oncology Diagnostics.

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly used material for tumor molecular profiling, therapy selection, and prognostication. Tumor tissue enrichment by tissue dissection is highly recommended to generate quality data reproducibly for use in downstream assays, such as real-time PCR and next-generation sequencing. The aim of this study was to evaluate the performance of the automated tissue dissection tool AVENIO Millisect System compared with a manual dissection method using 18 FFPE tissue specimens. The study assessed performance of these two methods with paraffinized and deparaffinized sections at 5- and 10-µm thickness as well as at low (5% to 10%) and high (>50%) tumor content. In addition, compatibility with various nucleic acid and protein extraction methods was assessed. Overall, dissection by Millisect resulted in statistically significantly higher yields of nucleic acids and protein compared with manual dissection (P = 0.00524). In downstream analysis on a statistically nonpowered sample set, EGFR mutation testing by PCR led to highly concordant results, and next-generation sequencing testing yielded significantly higher allelic frequencies when tissue was dissected by Millisect compared with manual scraping, demonstrating noninferiority of the automated method. In summary, the AVENIO Millisect System may replace manual labor and support automation of FFPE tumor tissue workflows in clinical molecular laboratories with high testing volumes with adequate validation.

Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry.

A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal-a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.