To begin, prepare 10 times concentrated citrate, target retrieval solution or TRS of pH six, according to the manufacturer's instructions. Dilute it to a ratio of 1:10 with deionized water. Then heat the solution in a plastic staining jar to 96 degrees Celsius.
Remove the slides from the slide rack and place them into a wet chamber. Next, pipette one or two drops of the autofluorescence-reducing agent onto each tissue sample and incubate the samples for five minutes. Stack the slides back into the slide rack, and rinse for one minute in 60%ethanol using upward and downward motions to remove the unused autofluorescence-reducing reagent.
Submerge the slide rack in deionized water for five minutes. Transfer the slides into a staining jar with preheated TRS Buffer for Antigen Retrieval. Incubate the slides for 10 minutes at 96 degrees Celsius in a water bath.
Remove the staining jar and let it cool to room temperature for approximately 60 to 90 minutes. Next, remove the TRS while keeping the slides in the jar. Rinse the slides twice with TRIS Buffered Saline containing 0.05%Tween at pH 7.4 for three minutes each to remove any remaining TRS residues.
Place the slides in a wet chamber. Gently wipe excess water from the slides using paper tissues, then encircle each sample using a hydrophobic barrier pen to prevent the antibody solution from running off.
