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

0.01 M Phosphate-buffered saline, pH 7.4 (PBS)

Paraformaldehyde (PFA)

Bovine serum albumin (BSA)

Anti-HS antibody, F58-10E4 (Seikagaku Corp., Tokyo, Japan) (Note 1)

Alexa Fluor^® 488 goat anti-mouse IgM, 2 mg/mL (Thermo Fisher Scientific, Waltham, MA, USA)

Anti-mouse IgM biotin conjugate antibody

4’,6-Diamino-2-phenylindole (DAPI) (Thermo Fisher Scientific)

VECTASHIELS mounting medium (Vector Laboratories Inc., Burlingame, CA)

30% H₂O₂

Vectastain ABC kit (Vector Laboratories Inc.)

3-3’-Diaminobenzidine (DAB) tetrahydrochloride

Fluorescence microscope, BZ-9000 (KEYENCE Corporation, Osaka, Japan)

Staining of cells

a.

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

b.

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

c.

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

d.

Wash the cover glass with PBS for 1 s once and then for 5 min three times. (Note 2)

e.

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

f.

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

g.

Transfer the cover glass to a humid box.

h.

Add 200 μL of the anti-HS antibody F58-10E4 (diluted 1:200 in 1% BSA/PBS) and incubate at 4°C overnight. (Note 3)

i.

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

j.

Wash the cover glass with 1% BSA/PBS twice.

k.

Transfer the cover glass to a humid box.

l.

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 (Note 3).

m.

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

n.

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

o.

Wash the cover glass with PBS for 1 s once and then for 5 min three times.

p.

Mount the cover glass using the mounting medium.

q.

Capture images using a fluorescent microscope (Figure 1).

Staining of tissues

a.

Soak a paraffin-embedded section on a glass slide in xylene (three times), ethanol (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 (Note 4).

b.

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

c.

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

d.

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

e.

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

f.

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

g.

Tap off the excess BSA/PBS solution.

h.

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°C overnight.

i.

Tap off the excess antibody solution.

j.

Soak the slide in PBS for 1 s once and then in three changes in PBS for 5 min each (Note 5).

k.

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.

l.

Tap off the excess secondary antibody solution.

m.

Soak the slide in PBS for 1 s once and then in three changes in PBS for 5 min each.

n.

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.

o.

Tap off the excess ABC solution.

p.

Soak the slide in PBS for 1 s once and then in three changes in PBS for 5 min each (Note 5).

q.

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.

r.

Soak the slide in PBS for 1 s to terminate the reaction and then in three changes in PBS for 5 min each.

s.

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

t.

Soak the slide in three changes in xylene for 10 min each.

u.

Dry the section in air.

v.

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

w.

Capture images using a microscope (Figure 2).

Seikagaku Corp. discontinued its research reagent business on Sept. 30, 2011. Anti-HS antibodies are available from several companies, including Cosmo Bio Co., Ltd., Tokyo, Japan. Authentic HS preparations can be purchased from Sigma-Aldrich.

To permeabilize the cells, they are treated with the lysis buffer (PBS containing 0.1% Triton X-100) for 5 min at room temperature after step 1d of Methods and then washed with PBS twice.

The antibody solution should be diluted just before use.

In the case of a frozen section, the section is soaked in acetone/methanol (1:1) for fixing and incubated at room temperature for 3 min. After the section has been dried in air, experiments can be performed from step 2d of Methods.

Be careful not to overdevelop the preparations. The incubation period can be changed.