pH-dependent antigen unmasking in paraformaldehyde-fixed tissue cryosections.
Fras1/Frem family of basement membrane proteins has been associated with the "bleb" phenotype in mouse and the Fraser syndrome in man. Fras1 and Frem2 proteins are known to be colocalized in all epithelial basement membranes during embryonic development. The functional significance of their colocalization has been demonstrated in the corresponding mouse mutants, where the absence of Fras1 results in complete depletion of Frem2 from epithelial basement membranes and vice versa. Nevertheless, under standard immunohistochemical procedures, we were able to detect Fras1, but not Frem2, in the basement membrane of adult mouse tail skin. After reevaluation of our protocol, we established 15-minute acidic buffer treatment to be of critical value upon Frem2 immunodetection, essentially operating as an antigen retrieval process. Testing more polyclonal antibodies revealed no negative effects, but rather reinforced the positive signal, rendering this technique suitable for incorporation to any standard immunohistochemical procedure.
Pavlakis
Evangelos
E
Department of Biology, University of Crete, Heraklion, Crete, Greece.
Chalepakis
Georges
G
D000818:Animals; D000941:Antigens / Q000276:immunology / Q000378:metabolism; D001485:Basement Membrane / Q000196:embryology / Q000276:immunology / Q000378:metabolism; D016616:Cryoultramicrotomy; D016326:Extracellular Matrix Proteins / Q000172:deficiency / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D005557:Formaldehyde; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D051379:Mice; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D011108:Polymers; D012867:Skin / Q000196:embryology / Q000276:immunology / Q000378:metabolism; D016707:Tissue Fixation
D000941:Antigens; D016326:Extracellular Matrix Proteins; C482018:Fras1 protein, mouse; C504802:Frem2 protein, mouse; D011108:Polymers; D005557:Formaldehyde; C003043:paraform