Histochemical analysis of cell surface glycans by peroxidase labeled lectin
Peroxidase staining method is widely used and a powerful technique to detect localization of proteins on cells and tissues, and has an advantage of long-time preservation of slides. This protocol is for a detection of specific carbohydrate structures with a peroxidase lectin by using a horseradish peroxidase (HRPO)-based detection system. The procedure relies on proper fixation of cells to retain cellular distribution of antigen and to preserve cellular morphology.
Tissues of interest
10% (v/v) neutral buffered formalin or 4% paraformaldehyde in PBS, pH 7.4
100%, 95%, 75% ethanol
0.01M Citrate buffer, pH6.0
Blocking solution (1% BSA in PBS)
HRPO-conjugated avidin-biotin complex (ABC)
AEC substrate solution
Clear nail polish
Paraffin embedding of the tissues
Immerse the tissue fragment in 10% neutral buffered formalin or 4% paraformaldehyde in PBS.
Incubate for 6 to 8 hr.
Immerse the tissue in 75% ethanol for 15 min.
Immerse the tissue in 95% ethanol for 15 min and then for 20 min.
Immerse the tissue in 100% ethanol for 20 min x 3 times.
Immerse the tissue in xylene 20 min x 3 times.
Embed in paraffin.
Section 5- 8- μm thick, and place the sections on poly-L-lysine-coated slides.
Incubate the slides in a dry oven at 60oC for 1hr.
Deparaffinaization and hydration of paraffin-embedded section
Immerse the slides in a Coplin jar containing xylene for 4 min for x 5 times.
Immerse the tissue in 100% ethanol twice for 3 min each.
Immerse the tissue in 95% ethanol twice for 3 min each.
Immerse the tissue in 75% ethanol twice for 3 min each.
Immerse slides in tap water for 5 min.
Immerse slides into citrate buffer (0.01M, pH6.0).
Microwave (700 W) for 5 min.
Immerse in cold PBS.
Wash the slides twice with PBS.
Blocking of the endogenous peroxidase
Incubate the slide in 0.3% H2O2/methanol for more than 20 min.
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 at 30 min.
Remove as much of the solution as possible by tilting the slides.
Apply biotinylated lectin in sufficient quantity to cover the tissue.
Incubate at room temperature for 1hr in the dark.
Wash the slides 5 min x 3.
Detection of bound lectin
Incubate the slide 20 min in HRPO-conjugated avidin-biotin complex (ABC).
Add AEC substrate solution to the slide and incubate 10 min.
Transfer the slide to a Coplin jar.
Wash 10 min in running tap water.
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 light microscope.
Fixed, washed coverslips can be stored in PBS at 4oC for several days before staining. Incubation with lectin can be extended to overnight at 4oC if necessary. Tissues can be counterstained with hematoxylin if necessary. If background staining with lectin is too high, the lectin solution may be diluted further, wash for longer time, or try higher concentration of BSA in blocking solution. If specific staining is observed, but it is very faint, it is possible to increase the concentration of the lectin solution.
Figure & Legends
Fig.1 Histochemical analysis of human kidney stained by peroxidase labeled MBP
The section of mouse kidney was stained with biotin conjugated human MBP followed by HRP labeled avidin. The section was also counterstained with hematoxylin. Magnification of the cortex indicates brush border membrane staining in proximal renal tubules and the lack of staining in renal corpuscles.
* This figure was originally published in J Immunol. Hirano M, Ma BY. et al. "Mannan-binding protein blocks the activation of metalloproteases meprin alpha and beta" 2005, 175(5):3177-85. © The American Association of Immunologists, Inc.
Copyright 2005. The American Association of Immunologists, Inc. for Fig.1 in Figure & Legends
Copyright 2010. Ritsumeikan University, JCGGDB & AIST. for the rest of the contents
Current protocols in immunology (Vol.5), John Wiley & Sons, Inc.
Shin-sensyokuhou (Medical Technology Sup.), Ishiyaku Publishers, Inc.