Immunofluorescence and glutaraldehyde fixation. A new procedure based on the Schiff-quenching method.
The immunofluorescence technique is one of the most useful methods for localizing antigens in several tissues, including the central nervous system. For immunohistochemical procedures, especially immunofluorescence methods, formaldehyde is commonly used as a fixative agent. But for some protocols, mainly in neurobiology, glutaraldehyde is necessary to recognize a number of small molecules (haptens) whose antisera have been raised using glutaraldehyde as the cross-linking agent. This is a severe limitation because glutaraldehyde gives rise to a strong autofluorescence on tissue that precludes the observation of specific immunofluorescence staining. In this paper we present a new method that allows the use of immunofluorescence techniques on glutaraldehyde-fixed tissues. The new method consists of a treatment of tissue sections with the Schiffs reagent (leucobasic fuchsin) followed by a reduction of the Schiff-dye with sodium borohydride. This reduced dye produces a quenching of glutaraldehyde-induced fluorescence on the tissue. The goal of the new method is to make possible the use of a great number of available glutaraldehyde-raised antisera for immunofluorescence techniques, a useful tool in both basic and clinical research.
Tagliaferro
P
P
Instituto de Biología Celular y Neurociencias Prof. Eduardo De Robertis, Facultad de Medicina, Universidad de Buenos Aires, Argentina.
Tandler
C J
CJ
Ramos
A J
AJ
Pecci Saavedra
J
J
Brusco
A
A
D000818:Animals; D000906:Antibodies; D001894:Borohydrides; D004396:Coloring Agents; D003432:Cross-Linking Reagents; D016650:Fluorescein-5-isothiocyanate; D019085:Fluorescent Antibody Technique, Direct / Q000592:standards; D005904:Glial Fibrillary Acidic Protein / Q000276:immunology; D005976:Glutaral / Q000737:chemistry; D007202:Indicators and Reagents; D008297:Male; D008636:Mesencephalon / Q000737:chemistry / Q000166:cytology; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D013194:Staining and Labeling; D013438:Sulfhydryl Compounds; D016707:Tissue Fixation / Q000379:methods*
D000906:Antibodies; D001894:Borohydrides; D004396:Coloring Agents; D003432:Cross-Linking Reagents; D005904:Glial Fibrillary Acidic Protein; D007202:Indicators and Reagents; D011506:Proteins; D013438:Sulfhydryl Compounds; C025364:sodium borohydride; D016650:Fluorescein-5-isothiocyanate; C005732:thioacetic acid; D005976:Glutaral