Adrenal Tissue Autofluorescence Quenching

Frozen mouse adrenal tissue sections were thawed for 30 min at RT and washed with PBS two times, 10 min each, to remove O.C.T. The tissue sections were then treated separately with tissue AF treatment methods (Table 1) at room temperature. Sudan Black B (SBB, Dia-m, Moscow, Russia) was prepared as 0.1% (W/V) in 70% ethanol, as described previously [27 (link)]. Sections were incubated with the SBB solution sealed airtight in the dark for 20 min, and then dipped briefly in 70% ethanol once before washing with PBS. A solution of 10 mM copper(II) sulfate (CuSO₄) in 50 mM ammonia acetate, pH 5, was prepared and applied to sections for 90 min [27 (link)]. Ammonia/ethanol (NH₃) was prepared as 0.25% (V/V) ammonia (PanReac AppliChem ITW Reagents, Barcelona, Spain) in 70% ethanol and applied to tissue sections for 1 h [29 (link)]. A fresh 0.05% (W/V) trypan blue (Paneko, Moscow, Russia) in PBS solution was prepared and applied to slides for 15 min [28 (link)]. The 20X TrueBlack solution (TrueBlack^TM Lipofuscin Autofluorescence Quencher, Biotium, Fremont, CA, USA) was diluted to 1X in 70% ethanol and applied to tissue sections for 1 min. After each of these treatments, the slides were washed with PBS 3 times for 15 min each. TrueVIEW Reagent (TrueVIEW^TM Autofluorescence Quenching Kit, Vector Laboratories, Burlingame, CA, USA) was prepared according to manufacturer instructions, applied immediately to sections for 3 min, and then washed once with PBS for 5 min. MaxBlock^TM Autofluorescence Reducing Reagent Kit (MaxVision Biosciences, Bothell, WA, USA) was applied according to manufacturer instructions. Tissue sections were incubated with MaxBlock^TM Autofluorescence Reducing Reagent (Reagent A) for 1 min, washed according to manufacturer instructions, incubated with Post-Detection Conditioner (Reagent B) for 5 min, and then washed according to manufacturer instructions. After each treatment, the slides were mounted onto coverslips with a polyvinyl alcohol mounting medium with DABCO^TM antifading (Sigma-Aldrich, St. Louis, MO, USA). The slides treated with TrueVIEW^TM Autofluorescence Quenching Kit were mounted with VECTASHIELD Vibrance Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. The slides were stored at 4 °C in the dark for subsequent examination.