Application of anti-GAG antibody and biotinylated hyaluronan binding protein(bHABP) [3] ~ Immunostaining using anti-heparan sulfate antibody

 Seed cells on a cover glass in a 6-cm dish.

 Transfer the cover glass to a 6-well plate and wash it with PBS three times.

 Add 1 mL of 4% PFA/PBS to the well for fixing and incubate at room temperature for 30 min.

 Wash the cover glass with PBS for one second once and then for 5 min three times.

 Add 200 μL of 1% BSA/PBS to the cover glass for blocking and incubate at room temperature for 30 min.

 Wash the cover glass with PBS two times and then with 1% BSA/PBS once.

 Transfer the cover glass to a humid box.

 Add 200 μL of the anti-HS antibody F58-10E4 (diluted 1:200 in 1% BSA/PBS), and incubate at 4^oC overnight.

 Transfer the cover glass to a 6-well plate and wash it with PBS for 5 min three times.

 Wash the cover glass with 1% BSA/PBS two times.

 Transfer the cover glass to a humid box.

 Add 200 μL of the secondary antibody, Alexa Fluor^® 488 goat anti-mouse IgM (diluted 1: 200 in 1% BSA/PBS), and incubate for 1 h at room temperature with light shielding.

 Transfer the cover glass to a 6-well plate and wash it with PBS for 5 min three times.

 Add 1 mL of 5 ng/mL DAPI and incubate for 5 min at room temperature with light shielding.

 Wash the cover glass with PBS for one second once and then for 5 min three times.

 Mount the cover glass using the mounting medium.

 Capture images with a fluorescent microscope (Fig. 1).

 Soak a paraffin-embedded section on a glass slide in 100% (twice), 90%, 70%, and 50% ethanol sequentially for 5 min each, and then in 2 changes of H₂O for 5 min each in a Coplin jar at room temperature for deparaffinization and hydration.

 Soak the section in 2 changes of PBS for 5 min and then 0.1% Triton X-100/PBS for 10 min for retrieval of the antigen.

 Soak the slide in 2 changes of PBS and then in H₂O for 5 min.

 Soak the slide in 3% H₂O₂/methanol at room temperature for 30 min for inactivation of endogenous peroxidases.

 Soak the slide in 2 changes of H₂O and then 2 changes of PBS for 5 min each.

 Add 200 μL of 3% BSA/PBS to the section for blocking in a humid box and incubate for 1 h at room temperature.

 Tap off the excess BSA/PBS solution.

 Add 200 μL of the anti-HS antibody, F58-10E4 (diluted 1:100 in 1% BSA/PBS), to the section in a humid box and incubate at 4^oC overnight.

 Tap off the excess antibody solution.

 Soak the slide in PBS for one second once and then in 3 changes of PBS for 5 min each.

 Add 200 μL of the secondary antibody, anti-mouse IgM biotin conjugate antibody (diluted 1:100 in 1% BSA/PBS), to the section in a humid box and incubate at room temperature for 1 h.

 Tap off the excess secondary antibody solution

 Soak the slide in PBS for one second once and then in 3 changes of PBS for 5 min each

 Add 200 μL of the ABC solution of the Vectastain ABC kit (diluted 1:200 in PBS) to the section in a humid box and incubate at room temperature for 1 h

 Tap off the excess ABC solution.

 Soak the slide in PBS for one second once and then in 3 changes of PBS for 5 min each.

 Soak the slide in 0.06% DAB/PBS for 10 min and then in 0.01% H₂O₂/0.06% DAB/PBS within 10 min until the color is developed.

 Soak the slide in PBS for one second to terminate the reaction and then in 3 changes of PBS for 5 min each.

 Soak the slide in H₂O for 5 min and then in 20%, 40%, 80%, and 100% ethanol sequentially for 5 min each.

 Soak the slide in 3 changes of xylene for 10 min each.

 Dry the section in air.

 Mount the sections under a cover glass using the mounting medium.

 Capture images with a microscope.