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The sections were rehydrated by placing the slides in three changes of xylene for 10 min each, then graded ethanol from 100 to 50%%, followed by water and Tris-buffered saline.
Cells were fixed by placing the slides in ethanol.
Following a series of TBS washes, antigen retrieval was performed by placing the slides in 1% SDS in TBS at 37°C for 20 minutes.
The probes, together with the sperm, were denatured by placing the slides on a slide warmer at 75°C for 3 minutes, and then incubated overnight at 37°C in a humidified chamber.
Prior to staining, heat-induced antigen retrieval was performed by placing the slides into target retrieval solution, high pH (DakoCytomation), and steaming them in a commercial vegetable steamer at full temperature for 30 minutes (CEBPD) or by pressure cooking in TRIS/Citrate (pH 6) for 8 minutes (alpha-smooth muscle actin, ACTA2).
After rehydration, antigen retrieval was achieved by placing the slides in 100°C citrate buffer, pH 6.4 for 50 min.
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It was an advantage for reading slides that the area where AFB had been concentrated using the magnetic workstation was clearly defined and visible which helped with placing the slide on the microscope stage.
The prehybridization buffer was rinsed off by placing the slide in a 50-ml tube for 2 min, first in H2O, then in 70% ethanol, and then in 100% ethanol.
The mount was rehydrated in increasing dilutions of ethanol in distilled water (70%, 50 %, 30 %, 10 0%, 10 minutes each) and then stained by placing the slide in Carmine Alum stain over night.
Place the slides in a colander.
12. Once frozen, place the slide in a pre-cooled, clean, small plastic slide box inside the cryo chamber.
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