Non-enzymatic retrieval of antigen permits staining of follicle centre cells by the rabbit polyclonal antibody to protein gene product 9.5.
The rabbit polyclonal antibody to protein gene product 9.5 (PGP9.5) will detect the L1 isoenzyme of ubiquitin carboxy-terminal hydrolase (UCH), which is a marker for neurones and neuroendocrine tissue. We re-evaluated this antibody using the technique of non-enzymatic antigen retrieval (boiling sections in citrate buffer, heated by microwave oven) followed by streptavidin-biotin-peroxidase staining. Due to the fortuitous choice of appendix as positive control material containing small nerves, we found strong, repeatable cytoplasmic and nuclear staining of lymphoid follicle centre cells in addition to neural tissue. This effect could be repeated on other lymphoid tissues and was not dependent on microwave heating, but did require boiling in an ionic buffer solution. Staining was also observed with a fresh batch of antibody and with four of the five different batches of antibody which were supplied to us. This pattern was not obtained in fresh tissue, in fixed material following trypsinization, or by increasing the primary antibody concentration. We suggest that the boiling of sections in citrate buffer is exposing an epitope for the anti-PGP9.5 antibody which is inaccessible in the native or fixed state and therefore we would recommend retesting of antibody specificity following non-enzymatic retrieval of antigen.
Langlois
N E
NE
University of Aberdeen, Scotland, U.K.
King
G
G
Herriot
R
R
Thompson
W D
WD
D000818:Animals; D000906:Antibodies; D000941:Antigens / Q000302:isolation & purification*; D001065:Appendix / Q000737:chemistry*; D006358:Hot Temperature; D007150:Immunohistochemistry; D008872:Microwaves; D014066:Palatine Tonsil / Q000737:chemistry; D011817:Rabbits; D013194:Staining and Labeling; D013869:Thiolester Hydrolases / Q000276:immunology*; D043222:Ubiquitin Thiolesterase
D000906:Antibodies; D000941:Antigens; D013869:Thiolester Hydrolases; D043222:Ubiquitin Thiolesterase