Histochemical analysis of cell surface glycans by fluoresceinated lectin

 Dissolve FITC in DMF at 10 mg/mL.

 Mix well to completely dissolve the FITC.

 Dissolve the lectin up to 1 mg in 0.5 mL of 50 mM borate buffer, pH 8.5.

 Add 15 to 20-fold molar excess of FITC to 0.5 mL of lectin solution and immediately mix it.

 Incubate for 1 h at room temperature in the dark.

 Remove excess and hydrolyzed FITC by dialysis.

 Store the fluoresceinated lectin at 4°C in the dark.

 Place the freshly isolated tissue in a petri dish containing PBS and precut piece of aluminum foil.

 Apply OCT embedding compound to the fragment sufficiently to completely cover the tissue.

 Fold the foil to create a secure envelope.

 Drop the foil envelope directly into liquid nitrogen.

 Incubate the specimen for 5 to 10 min.

 Open the aluminum foil envelope containing the tissue inside the cryostat.

 Place the tissue on the OCT-coated chuck in the appropriate orientation.

 Allow the tissue to freeze solid for 10 min at −20°C inside the cryostat.

 Set the cryostat to cut 3 to 8-μm-thick sections.

 Allow the glass slide with the cryostat sections to air dry for more than 30 min.

 Immerse the slides 5 min in a Coplin jar containing acetone or 4% PFA-PBS.

 Allow the slides to air dry 10 min at room temperature.

 Using a pen containing water-repellant wax, outline the tissue sections on the glass slide.

 Place the slide in a staining chamber.

 Add blocking solution to the slides.

 Incubate for 30 min.

 Remove as much of the solution as possible by tilting the slides.

 Apply fluoresceinated lectin in sufficient quantity to cover the tissue.

 Incubate at room temperature for 1 h in the dark.

 Wash the slides three times for 5 min each.

 Place 1 drop of mounting medium onto slide.

 Place the coverslip onto drop.

 Gently blot mounted coverslip with paper towel.

 Seal edge of coverslip onto slide by painting the edge with a rim of nail polish and let dry.

 View specimen on fluorescence microscope.