Effect of tissue processing on the immunostaining of inflammatory cell surface epitopes identified by monoclonal antibodies.
Author(s): Visa K
Publication: Histochemistry, 1986, Vol. 84, Page 11-4
PubMed ID: 2420754 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to identify optimal tissue processing for analysis of frozen tissue (tonsil, lymph node, skin) by immunohistochemistry (IHC) for cell surface epitopes. The authors compared IHC staining with and without a methanol-peroxide step to block endogenous peroxidases and report on (data not shown) comparisons between tissue specimens that were frozen in liquid nitrogen versus pre-cooled isopentane (-56°C), with and without OCT. The author also assessed immunostaining and shared anecdotal observations following different durations of frozen storage (-50°C).
Conclusion of Paper
Although data was not shown, the authors report that morphology was better preserved by freezing in isopentane (-56°C) than liquid nitrogen. The authors also report embedding skin tissue specimens in OCT compound before snap-freezing (in liquid nitrogen or pre-cooled isopentane) diminished the intensity of immunostaining intensity for all of the cell surface epitopes evaluated but attenuated desiccation of the tissue specimen when it was stored at -50°C for 4-8 weeks (data not shown). Although data was not shown, the authors report that immunostaining was comparable for all cell surface epitopes evaluated when slides of snap-frozen tissues were fixed in acetone for 5 min at either 4°C or -20°C.
Although immunopositive staining was observed for all epitopes at varying intensities without treatment, incubating slides in 0.3% 0.3% H2O2 in methanol to quench endogenous peroxidase activity abolished staining for all antibodies) with the exceptions of OKT6 (S-100, immunostaining reduced from +++ to +), OKIa1 (immunostaining reduced from +++ to ++), and Leu 7 (CD57, immunostaining intensity was unchanged).
Studies
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Study Purpose
The purpose of this study was to identify optimal tissue processing for analysis of frozen tissue (tonsil, lymph node, skin) by immunohistochemistry (IHC) for cell surface epitopes. The authors compared IHC staining with and without a methanol-peroxide step to block endogenous peroxidases and report on (data not shown) comparisons between tissue specimens that were frozen in liquid nitrogen versus pre-cooled isopentane (-56°C), with and without OCT. The author also assessed immunostaining and shared anecdotal observations following different durations of frozen storage (-50°C). Surgical biopsies of tonsil and lymph nodes were embedded in OCT compound and snap-frozen in liquid nitrogen or isopentane pre-cooled to -56°C and stored at -20°C. Skin biopsies from benign conditions (papules, epicutaneous skin reactions due to a nickel allergy, intradermal nuclear antigen test reactions associated with connective tissue disease) were snap-frozen in liquid nitrogen or isopentane pre-cooled to -56°C with or without OCT compound and stored at -50°C. Tissue sections (4-5 µm) were cut on a cryostat, mounted on gelatin-coated slides, air-dried, and fixed in acetone at -20°C for 10 min or at 4°C for 5-20 min. Endogenous peroxidase activity of slides was either quenched by incubation with 0.3% hydrogen peroxide in methanol for 5-20 min or slides were left untreated. Immunohistochemical staining was conducted using the avidin-biotin-peroxidase complex technique after slides were incubated with normal horse serum to reduce nonspecific binding. Positive immunostaining was visualized using the chromagen 3.3’-diaminobenzidine tetrahydrochloride (DAB), and slides were counterstained with hematoxylin. The intensity of immunopositive staining was scored on a scale of “negative” to +++. The following mononuclear antibodies were used to investigate the staining of cell surface antigens (specified in parentheses where possible): were investigated using the monoclonal antibodies specified: OKT3 (CD3), OKT4 (CD4), OKT6 (S-100), OKM1 (CD11b), pan B cell (Ig light chains), Leu7 (CD57), OKIa1.
Summary of Findings:
Although data was not shown, the authors report that morphology was better preserved by freezing in isopentane (-56°C) than liquid nitrogen. The authors also report that embedding skin tissue specimens in OCT compound before snap-freezing (in liquid nitrogen or pre-cooled isopentane) diminished the intensity of immunostaining intensity for all of the cell surface epitopes evaluated but attenuated desiccation of the tissue specimen when it was stored at -50°C for 4-8 weeks, but no data was shown. Although data was not shown, the authors report that immunostaining was comparable for all cell surface epitopes evaluated when slides of snap-frozen tissues were fixed in acetone for 5 min at either 4°C or -20°C.
Although immunopositive staining was observed for all epitopes at varying intensities without treatment, incubating slides in 0.3% 0.3% H2O2 in methanol to quench endogenous peroxidase activity abolished staining for all antibodies (epitopes)(antibodies) with the exceptions of OKT6 (S-100, immunostaining reduced from +++ to +), OKIa1 (immunostaining reduced from +++ to ++), and Leu 7 (CD57, immunostaining intensity was unchanged).
Biospecimens
Preservative Types
- OCT
- Frozen
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform Protein Immunohistochemistry Glycoprotein Immunohistochemistry Carbohydrate Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Cooling or freezing method/ rate Liquid nitrogen
Pre-cooled isopentane
Biospecimen Preservation Temperature of fixation/preservation 4°C
-20°C
Immunohistochemistry Specific Reaction solution 0.3% H2O2 in methanol
No treatment
Biospecimen Preservation Type of fixation/preservation OCT
Snap frozen