Triton X-100 pretreatment of LR-white thin sections improves immunofluorescence specificity and intensity.
The staining of intracellular antigenic sites in postembedded samples is a challenging problem. Deterioration of antigenicity and limited antibody accessibility to the antigen are commonly encountered on account of processing steps. In this study preservation of the antigen was achieved by fixing the tissues with mild fixatives, performing partial dehydration, and embedding in a low crosslinked hydrophilic acrylic resin, LR-White. Permeabilization of cell membranes with Triton X-100 is well documented but can affect some antigen conformations. We tested the effect of Triton X-100 on the ED1 antigen present in the lysosomal membrane of the macrophage in cell culture. The ED1 antigen in the lysosome was resistant to extraction by Triton X-100. Interestingly pretreating the LR-White sections of macrophage pellets with Triton X-100 improved the staining intensity of ED1. The most intense and clear specific fluorescent staining was observed when sections were pretreated with 0.2% Triton X-100 for 2 min. Longer exposure of sections to 0.2% Triton or 2 min exposure to 2% Triton lead to reduced ED1 labeling. SEM observations indicated that the detergent extracted a component from the cells and not the resin and was determined to be lipid. This novel technique could be applied in many research areas where postembedding fluorescent immunolabeling with higher labeling intensity is desired.
Ghrebi
Salem S
SS
Department of Oral Biological and Medical Sciences, Faculty of Dentistry, The University of British Columbia, Vancouver, British Columbia, Canada.
Owen
Gethin Rh
GR
Brunette
Donald M
DM
D000180:Acrylic Resins; D000818:Animals; D002460:Cell Line; D016616:Cryoultramicrotomy; D005455:Fluorescent Antibody Technique / Q000379:methods*; D007150:Immunohistochemistry; D008264:Macrophages / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D008867:Microtomy; D017830:Octoxynol; D016613:Plastic Embedding / Q000379:methods; D011309:Preservation, Biological / Q000379:methods; D051381:Rats; D012680:Sensitivity and Specificity
D000180:Acrylic Resins; D017830:Octoxynol; C048707:LR white