Immunocytochemical Characterization of Human Blood Cells

Abstract

A simple immunocytochemical method using calf intestinal alkaline phosphatase as the enzymatic indicator to demonstrate surface antigens on human blood cells has been developed. The blood cells were labeled with cell specific monoclonal antibodies followed by linkage with an antiimmunoglobulin alkaline phosphatase conjugate. Cytochemical demonstration of alkaline phosphatase activity on the blood cells reflects the presence of surface antigens on these cells. The effects on the cytochemical reaction of fixation, substrates, couplers, activators and inhibitors, and storage of cytologic materials have been examined systematically. The best staining conditions are to incubate labeled smears in a 0.04 M barbital buffer at pH 7.6 containing 30 mg% naphthol AS-TR phosphate, 40 mg% fast red ITR, and 1 mM levamisole. This method is both sensitive and specific and appears most practical for objective identification of the human blood cells.

Selective inactivation of NF-κB in the liver using NF-κB decoy suppresses CCl4-induced liver injury and fibrosis

“…Infiltration by neutrophils in the liver was also estimated by means of naphthol AS-D chloroacetate esterase staining (41). The number of esterase-positive polymorphonuclear cells was counted in 10 highpower fields (ϫ400) in each sample, and mean values were calculated.…”

New Developments in Immunochemistry with Immunoalkaline Phosphatase Methods

“…These are direct, indirect, alkaline phosphatase-antialkaline phospha tase (APAAP) and variations of an avidinbiotin complex (ABC-AP) system (table I) [7][8][9][20][21][22][23][24], In the direct method the pri mary antibody reacting with the antigen is directly linked with the enzyme indicator. This method is seldom used because of its low sensitivity and also because it requires that each primary antibody be conjugated to the enzyme.…”

“…This method is seldom used because of its low sensitivity and also because it requires that each primary antibody be conjugated to the enzyme. In the indirect method the en zyme indicator is linked to antibodies (sec ondary antibodies) specific to the immuno globulin of the animal species in which the primary antibodies are produced [7,8,20]. This method is more versatile and sensitive than the direct method.…”

“…The major concern here is pres ervation of cell morphology and sensitivity of staining. Of the several fixatives we have examined [8,20], a buffered formalin-ace tone mixture is the most satisfactory [8,20]. The addition of 0.1% Triton X-100 to this fixative not only will decrease nonspecific staining, but also will enhance penetration of reagents into the cells and therefore increase staining intensity [28].…”

“…7-10) [7-9, 13, 20, 22, 31, 33, 35, 36, 51-72], The APAAP method is most suita bly used for studies in smears and imprints ( fig. 9, 10) [9, 13, 51-58], although indirect methods also have been used with success in cell suspensions and smears [8,13,20,21,[59][60][61][62][63][64][65], The ABC-AP methods are most suit able for the in situ hybridization studies [24,25,36], The objectives of these immunocytochemical studies are for characterization Fig. 8.…”