The effect of fixatives and paraffin embedding on the histochemical reactivity of a monoclonal antibody against human type IV collagen (JK-199).
A new monoclonal antibody (JK-199) was found to react with basement membranes on paraffin-embedded tissue sections without prior enzyme digestion. JK-199 was shown to react with isolated type IV collagen treated by any of four different fixatives--PLP, 4% formalin, modified Zamboni's (0.2% picric acid, 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) or Bouin's--applied for 6 h at room temperature and incubated at 60 degrees C for 30 min to simulate routine tissue processing. None of the fixatives was able to alter the reactivity of JK-199 with isolated type IV collagen. In the human placenta, specific and intense staining of basement membranes was demonstrated on paraffin sections fixed with any of the four fixatives. In human skin, basement membranes were fully demonstrated on paraffin sections fixed by PLP fixative or by 4% formalin, but only partially on sections fixed by picric acid-containing fixatives. Optimal results, i.e., with the least non-specific or incomplete staining, were obtained on PLP-fixed paraffin-embedded tissues. In PLP-fixed paraffin sections of the kidney, skeletal muscle, and small intestine, all basement membranes were stained intensely by this antibody (JK-199) at the expected locations. The results indicate that JK-199 may be widely applicable for the analysis of basement membrane kinetics, including developmental processes or pathological conditions.
Adachi
E
E
Department of Anatomy, Osaka University Medical School, Japan.
Kino
J
J
Hayashi
T
T
Sato
Y
Y
Hashimoto
P H
PH
D000911:Antibodies, Monoclonal; D003094:Collagen / Q000276:immunology*; D005404:Fixatives; D006801:Humans; D007158:Immunologic Techniques; D008867:Microtomy; D010232:Paraffin; D010920:Placenta / Q000166:cytology / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D005404:Fixatives; D010232:Paraffin; D003094:Collagen