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Fig. 2. Equipment required for free-fl oating sections immunohistochemistry. (1) Pipettor stand and (2) Glass slides. There
are no special brand preferences. However, slides should be compatible for use in fl uorescent microscopy. For example,
VWR Micro slides Superfrost Plus, VWR cat. No 48311-703, (3) Plates for tissue wash and incubation. Multiple Well Cluster
Plate, 24 Well, Tissue Culture-Treated, Corning Costar #3526, (4) Black tray helps visualize slides while washing and
transfer between plates. (5) Paint brushes to pick up tissue sections, (6) Syringe fi lters Millex-FG, 0.20
μ
m, hydrophobic
PTFE, 4 mm, cat. #SLFGR04NL, (7) Vectashield mounting media. If mounting media is not a hard set, a small amount of
nail polish can be applied on the cover slip edges to prevent its slipping. (8) Cover slips. It is critical to ensure that cover
slips can be used for fl uorescence. As example, Fisher scientifi c, Fisherfi nest Premium cover glass, #12548B, (9) Gooseneck
lamp, (10) Sterile tips, (11) Clear nail polish, (12) Serum. To avoid multiple freezing-thawing cycles, serum can be aliquoted
into 2-ml tubes and saved at −20°C until usage, (13) Incubator buffer. All tubes and tips require sterilization to prevent
contamination. (14) Ice container, (15) Wash buffer. During the procedure, all solutions and chemicals should be kept on
ice, (16) Antibodies. Antibody conjugated with fl uorescent dyes need to be light protected, (17) For negative control sec-
tions use a separate 24-well plate to avoid contamination, (18) Rotator. For overnight immunostaining, another rotator
should to be placed into the refrigerator. (19) Water bath. Some antibody retrieval protocols require in water bath or micro-
wave incubation. (20) During washing or incubation with antibodies containing a fl uorescent dye, the plate should be
covered with aluminum foil.
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