TY - JOUR
T1 - Selection of a fixative for identifying T cell subsets, B cells, and macrophages in paraffin-embedded mouse spleen
AU - Gendelman, Howard E.
AU - Moench, Thomas R.
AU - Narayan, Opendra
AU - Griffin, Diane E.
N1 - Funding Information:
The authors wish to thank Dr. William Stroop, University of Pennsylvania, for helpful suggestions of fixation techniques, Dr. William Mobley, Johns Hopkins University, for suggestions with fixation and embedding procedures, Ms. Elaine Langlois and Linda Kelly for secretarial assistance, Dr. Jonathan Austyn for the gift of antibody F4/80, Dr. Robert Coffman for the gift of antibody RA32C2, and Diane Stewart and Querubin P. Mendoza for technical assistance. This study was supported in part by Public Health Service Grants NS-15721, NS-18596 and NS-07000 from the National Institutes of Health.
PY - 1983/12/16
Y1 - 1983/12/16
N2 - Fixation techniques were investigated for their ability to preserve morphology, esterase activity and cell surface antigens in paraffin-embedded mouse lymphoid tissue. The avidin-biotin-peroxidase system was used to stain antigens Thy-1.2, Lyt-1, Lyt-2, RA32C2 and F4/80. Conventional fixatives were compared with fixatives containing periodate and lysine plus paraformaldehyde and/or glutaraldehyde. Conventional fixatives preserved esterase activity but not many cell surface antigens. Periodate-lysine fixatives allowed better preservation of membrane antigens, but esterase activity was often lost at antigen-preserving concentrations of paraformaldehyde or glutaraldehyde. However, a periodate-lysine fixative containing both paraformaldehyde and glutaraldehyde preserved esterase and showed good to excellent staining of Lyt-1, Thy-1.2, RA32C2, and F4/80. Lyt-2 could not be stained with any fixative, but was well preserved in frozen material post-fixed with periodate-lysine based fixatives. We conclude that with proper fixation immune cell surface markers can be identified in paraffin-embedded tissue.
AB - Fixation techniques were investigated for their ability to preserve morphology, esterase activity and cell surface antigens in paraffin-embedded mouse lymphoid tissue. The avidin-biotin-peroxidase system was used to stain antigens Thy-1.2, Lyt-1, Lyt-2, RA32C2 and F4/80. Conventional fixatives were compared with fixatives containing periodate and lysine plus paraformaldehyde and/or glutaraldehyde. Conventional fixatives preserved esterase activity but not many cell surface antigens. Periodate-lysine fixatives allowed better preservation of membrane antigens, but esterase activity was often lost at antigen-preserving concentrations of paraformaldehyde or glutaraldehyde. However, a periodate-lysine fixative containing both paraformaldehyde and glutaraldehyde preserved esterase and showed good to excellent staining of Lyt-1, Thy-1.2, RA32C2, and F4/80. Lyt-2 could not be stained with any fixative, but was well preserved in frozen material post-fixed with periodate-lysine based fixatives. We conclude that with proper fixation immune cell surface markers can be identified in paraffin-embedded tissue.
KW - cell surface antigen
KW - immunoperoxidase staining
KW - monoclonal antibody
KW - paraffin embedding
KW - periodate-lysine-paraformaldehyde-glutaraldehyde
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U2 - 10.1016/0022-1759(83)90310-1
DO - 10.1016/0022-1759(83)90310-1
M3 - Article
C2 - 6317754
AN - SCOPUS:0021042295
SN - 0022-1759
VL - 65
SP - 137
EP - 145
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -