title: Immunohistochemical detection of T-cell subsets and other leukocytes in paraffin-embedded rat and mouse tissues with monoclonal antibodies.
abstract: We describe a method for immunohistochemical localization of T-cells, CD4+ T-cells, CD8+ T-cells, B-cells, activated lymphocytes, major histocompatibility complex (MHC) class II antigens, macrophages, dendritic cells, and granulocytes in rat and mouse tissue fixed in periodate-lysine-paraformaldehyde (PLP) and embedded in paraffin. Rat and mouse spleen and eyes were fixed in PLP for 18-24 hr, rapidly dehydrated, infiltrated under vacuum with paraffin at 54 degrees C, sectioned, and stained with appropriate monoclonal antibodies (MAbs). Sections of PLP-fixed, paraffin-embedded spleen were compared with acetone-fixed frozen spleen sections with respect to morphology and staining quality. Nine of 10 MAbs to rat antigens and eight of nine MAbs to mouse antigens stained paraffin sections equally or more intensely than frozen sections. The two MAbs that showed weaker staining still gave good staining on paraffin sections. Paraffin-embedded rat and mouse eyes were easier to section serially than frozen eyes, showed superior morphology, and individually stained cells were readily identified. Therefore, a combination of PLP fixation and low-temperature paraffin embedding permits detection of the major types of immune cell in rat and mouse tissues while maintaining good morphology, particularly in diseased, damaged, or delicate tissues.
authors.affiliation: Department of Ophthalmology, University of Bristol, United Kingdom.
authors.forename: J L
authors.initials: JL
authors.lastname: Whiteland
authors.forename: S M
authors.initials: SM
authors.lastname: Nicholls
authors.forename: C
authors.initials: C
authors.lastname: Shimeld
authors.forename: D L
authors.initials: DL
authors.lastname: Easty
authors.forename: N A
authors.initials: NA
authors.lastname: Williams
authors.forename: T J
authors.initials: TJ
authors.lastname: Hill
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D018845:CD11 Antigens / Q000032:analysis; D017252:CD3 Complex / Q000032:analysis; D015704:CD4 Antigens / Q000032:analysis; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D016827:CD8 Antigens / Q000032:analysis; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D007150:Immunohistochemistry / Q000379:methods; D017493:Leukocyte Common Antigens / Q000032:analysis; D007962:Leukocytes / Q000166:cytology*; D051379:Mice; D051381:Rats; D015375:Receptors, Interleukin-2 / Q000032:analysis; D012680:Sensitivity and Specificity; D016176:T-Lymphocyte Subsets / Q000166:cytology* / Q000276:immunology; D018800:Thy-1 Antigens / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D018845:CD11 Antigens; D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; D015375:Receptors, Interleukin-2; D018800:Thy-1 Antigens; D017493:Leukocyte Common Antigens