Seven-colour multiplex immunochemistry/immunofluorescence and whole slide imaging of frozen sections.

Multiplex Immunochemistry/Immunofluorescence (mIHC/IF) aims to visualise multiple biomarkers in a single tissue section and is especially powerful when used on slide scanners coupled with digital analysis tools. mIHC/IF is commonly employed in immuno-oncology to characterise features of the tumour microenvironment (TME) and correlate them with clinical parameters to guide prognostication and therapy. However, mIHC/IF can be applied to a wide range of organisms in any physiological or disease context. Recent innovation has extended the number of markers that can be detected using slide scanners well beyond the 3-4 markers typically reported in traditional fluorescence microscopy. However, these methods often require sequential antibody staining and stripping, and are not compatible with frozen tissue sections. Using fluorophore-conjugated antibodies, we have established a simple mIHC/IF imaging workflow that enables simultaneous staining and detection of seven markers in a single section of frozen tissue. Coupled with automated whole slide imaging and digital quantification, our data efficiently revealed the tumour-immune complexity in metastatic melanoma. Computational image analysis quantified the immune and stromal cell populations present in the TME as well as their spatial interactions. This imaging workflow can also be performed with an indirect labelling panel consisting of primary and secondary antibodies. Our new methods, combined with digital quantification, will provide a valuable tool for high-quality mIHC/IF assays in immuno-oncology research and other translational studies, especially in circumstances where frozen sections are required for detection of particular markers, or for applications where frozen sections may be preferred, such as spatial transcriptomics.

Purinergic Signaling and Aminoglycoside Ototoxicity: The Opposing Roles of P1 (Adenosine) and P2 (ATP) Receptors on Cochlear Hair Cell Survival.

Purinergic signaling regulates important physiological processes and the homeostatic response to stress in the cochlea via extracellular nucleosides (adenosine) and nucleotides (ATP, UTP). Using a previously established organotypic culture model, the current study investigated the effect of purinergic P1 (adenosine) and P2 (ATP) receptor activation on the survival of the sensory hair cell population in the cochlea exposed to the ototoxic aminoglycoside neomycin. Organ of Corti explants were obtained from C57BL/6 mice at postnatal day 3 (P3) and maintained in normal culture medium (with or without purine receptor agonists or analogs) for 19.5 h prior to neomycin exposure (1 mM, 3 h) followed by a further incubation for 19.5 h in culture medium. The cochlear explants were then fixed in 4% paraformaldehyde (PFA) and sensory hair cells labeled with Alexa 488-phalloidin. Neomycin induced a substantial loss of the sensory hair cells, mostly in the middle segment of the cochlea. This neomycin-induced ototoxicity was unaffected by the addition of P2 receptor agonists (ATP and UTP) in the culture medium, whilst the addition of their slowly-hydrolyzable analogs (ATPγS, UTPγS) aggravated neomycin-induced sensory hair cell loss. In contrast, the activation of P1 receptors by adenosine or adenosine amine congener (ADAC) conferred partial protection from neomycin ototoxicity. This study demonstrates a pro-survival effect of P1 receptor stimulation, whilst prolonged activation of P2 receptors has an opposite effect. Based on these findings, we postulate that P1 and P2 receptors orchestrate differential responses to cochlear injury and that the balance of these receptors is important for maintaining cochlear homeostasis following ototoxic injury.

Resistance to neomycin ototoxicity in the extreme basal (hook) region of the mouse cochlea.

Aminoglycoside ototoxicity results in permanent loss of the sensory hair cells in the mammalian cochlea. It usually begins at the basal turn causing high-frequency hearing loss. Here we describe previously unreported resistance of hair cells to neomycin ototoxicity in the extreme basal (hook) region of the developing cochlea of the C57BL/6 mouse. Organ of Corti explants from mice at postnatal day 3 were incubated (37 °C, 5% CO2) in normal culture medium for 19.5 h prior to and after exposure to neomycin (1 mM, 3 h). To study neomycin uptake in the hair cells, cochlear explants were incubated with Neomycin Texas-red (NTR) conjugate. As expected, exposure to neomycin significantly reduced the survival of inner (IHC) and outer hair cells (OHC). IHC survival rate was high in the apical segment and low in the basal segment. OHC were well preserved in the apical and hook regions, with substantial OHC loss in the basal segment. The NTR uptake study demonstrated that the high survival rate in the extreme basal turn OHC was associated with low NTR uptake. Treatment with a calcium chelator (BAPTA), which disrupts the opening of mechanoelectrical (MET) transduction channels, abolished or reduced NTR uptake in the hair cells throughout the cochlea. This confirmed the essential role of MET channels in neomycin uptake and implied that the transduction channels could be impaired in the hook region of the developing mouse cochlea, possibly as a result of the cadherin 23 mutation responsible for the progressive deafness in C57BL/6 mice.