Sudan Black B

Embedding procedures were performed on 100-μm brain slices, which were dehydrated in a graded ethanol series (50, 70, and 95% ethanol, changing from one concentration to the next every 5 min at 4°C). After dehydration, the brains were immersed in a graded GMA series (Ted Pella, United States), including 0.2% Sudan black B (SBB)(Sun et al., 2011 (link)) (70, 85, and 100% GMA for 15 min each and 100% GMA overnight at 4°C). For whole-brain embedding, brains were dehydrated in a graded ethanol series every 1 h at 4°C and immersed in a graded GMA series for 2 h each and 100% GMA overnight at 4°C. Subsequently, the samples were impregnated in a prepolymerization GMA solution for 3 days at 4°C and embedded in a vacuum oven at 35°C for 24 h. The 100% GMA solution comprised 52.5 g of A solution, 2.8 g of deionized water, 44.1 g of B solution, 0.2 g of SBB, and 0.8 g of ABVN as an initiator. The 70 and 85% GMA solutions (wt/wt) were prepared from 95% ethanol and 100% GMA.

For cell immunofluorescence, 2 × 10⁴ cells were seeded on each confocal dish and cultured for 1 day. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature. After permeabilizing with 0.1% Triton X-100 or cold digitonin (Abcam) for 10 min and blocking with normal goat serum for 30 min, samples were incubated with primary antibodies overnight at 4 °C, including anti-TOM20 (clone F-10, Santa Cruz Biotechnology; BM4366, Boster), anti-PD-L1 (clone EPR19759, Abcam; LS-C754760, LifeSpan BioSciences; 2B11D11, Proteintech), anti-LAMP1 (clone D2D11, Cell Signaling Technology; clone 1D4B, Santa Cruz Biotechnology), anti-TGN46 (clone 1F6D5, Proteintech), anti-Calnexin (clone 2A2C6, Proteintech). The cells were washed with PBS-T for three times and incubated with secondary antibodies (Alexa Fluor Plus 555, Alexa Fluor Plus 488, Alexa Fluor Plus 647, Invitrogen) at room temperature for 1 h. Antifade Mounting Medium with DAPI (Beyotime) was used to mount the samples. The confocal images were then taken on Zeiss LSM 900 and Airyscan 2 confocal laser scanning microscope and analyzed with ZEN 2.3 software.
For MitoTracker staining, cells were incubated with MitoTracker Deep Red dyes in complete medium at 37 °C for 30 min, then were removed of excess unbound dyes by washing twice.
For tissue immunofluorescence, paraffin sections of patients with TNBC were baked at 60 °C for 30 min followed by deparaffinization and rehydration using xylene and graded ethanol. After EDTA (pH 8.0) antigen retrieval and endogenous peroxidase blockade as IHC staining methods mentioned above, the samples were permeabilized with 0.3% Triton for 30 min and blocked with normal goat serum for 30 min at room temperature. Primary antibodies targeting PD-L1, LAMP1 and TOM20 were incubated with samples overnight at 4 °C. After secondary antibody incubation for 1 h and DAPI incubation for 15 min, 0.3% Sudan black-B solution was used to quench spontaneous fluorescence for 20 min at room temperature followed by washing twice. The confocal images were then taken and analyzed.

Isolation and detection for PHA producers using lipophilic stain Sudan black B is a presumptive test [16 (link)]. Stain was prepared by dissolution of 0.3 g of dye in 100 ml of 70% ethanol. Stain was flooded on the colonies in selective medium; after 30min, the excess dye was decanted and washed with 100% ethanol, and colonies appear bluish black and accredited as PHA-positive strains.

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.