Sources/Clones
Accurate (84B3-1), Biodesign (RB1, 1F8), Biogenesis (RB), Biomol Research (MAB245), Dako (Rb1), Labvision Corp (1F8), Novocastra (Rb1), Oncogene (AF11, LM95.1), Pharmingen (245), QED (3C8) and Santa Cruz (C-15)
Fixation/Preparation
The antibodies are mostly immunoreactive only in fresh-frozen sections although some antibodies stain fixed paraffin-embedded sections but only after HIER.
Background
The Rb gene is located on chromosome 13q14 and spans a region of more than 200 kb, including 27 exons. The Rb gene is the only tumor suppressor that has been shown to directly suppress tumor formation. The Rb protein has a molecular mass of 105 kD and a number of antibodies which recognize specific parts of this protein have been developed. Besides loss of function due to chromosomal abnormalities including chromosomal deletion, translocation and point mutation, as with p53, phosphorylation may inactivate the Rb protein. In addition, a variety of viral oncoproteins including simian virus 40 T antigen, E1A from adenovirus and E6 from human papilloma virus may bind and inactivate the Rb protein. Immunostaining may be a valid way to assess the presence of normal Rb protein but several factors affecting staining should be considered before accepting the relevance of the technique. Firstly, it has been observed that the level of expression of Rb protein is not the same in all cells in any individual tissue, e.g. in the epithelium of the cervix, there are low or undetectable levels of staining in the basal layers and staining increases with cell maturation. In contrast, low or absent anti-Rb protein staining was observed in the well-differentiated epithelial cells of the gastric mucosa such as the foveolar and mucus cells compared to the cells in the crypts and neck of the glands. Astrocytes and microglia do not show detectable Rb protein by immunostaining and other subsets of normal cells such as some stromal cells do not display demonstrable Rb protein. The reasons for failure to demonstrate the protein at an equivalent level in all cells may relate to variations in expression as a function of cell cycling activity, cell differentiation and protein phosphorylation. More importantly, there is a large subset of cells, which RB include endothelial cells, lymphocytes and stromal cells, in which the ability to demonstrate p110expression is critically dependent on the method of staining used (Cordon-Cardo & Richon, 1994).
Applications
The p53 and retinoblastoma (Rb) gene products must be the two most-studied tumor suppressor genes. While alterations in the p53 tumor suppressor gene have been recognized as the most frequent genetic alterations in human neoplasia, the extent of Rb gene alterations is less well known. p53 alterations are mostly detected as overexpression of the protein and can easily be done with immunostaining, whereas most normal cells do not contain stainable wild-type p53 protein. In contrast, the Rb protein is detectable immunohistochemically in normal non-transformed cells, although whether this is so for all normal cells and tissues is currently unknown. As abnormality is based on the absence of stainable Rb protein, it is critical that techniques of maximal sensitivity must be employed and internal positive controls must be present in the sections (Skelly et al, 1996).
Alterations in the RB gene have been described in a number of human tumors including retinoblastoma, osteosarcoma, other sarcomas, leukemias, lymphomas and certain carcinomas including those from the breast, prostate, lung, bladder, kidney and testis (Geradis et al, 1994). Rb gene alterations have been associated with increasing tumor grade and stage in a variety of tumors and there is increasing evidence that alterations of this gene are associated with increased risk for metastasis (Xu et al, 1991, 1993). In breast carcinoma there is some evidence of association with other signs of progression and loss of hormonal receptor expression (Drobnak et al, 1993).
Comments
It was recently demonstrated that HIER in citrate buffer at pH 6.0 with overnight antibody incubation produced maximal sensitivity when staining fixed paraffin-embedded sections. Fixation in methacarn also requires HIER treatment and the use of DNAse produced variable results. The use of low pH buffers can produce false-positive results. Thus, in the assessment of Rb protein, as with other fixation-sensitive antigens, it is clear that the findings of individual laboratories cannot be generalized owing to differences in fixation and immunolabeling techniques. However, these factors do not preclude the assessment of the Rb protein in laboratories where fixation and other variables are strictly controlled.
References
•Cordon-Cardo C, Richon VM 1994. Expression of the retinoblastoma protein is regulated in normal human tissues. American Journal of Pathology 144: 500-510.
•Drobnak M, Cote RJ, Saad AD et al 1993. P53 and Rb alterations in primary breast carcinoma: correlation with hormone receptor expression and lymph node metastases. International Journal of Oncology 2: 173-178.
•Geradis J, Hu SX, Lincoln CE et al 1994. Aberrant RB gene expression in routinely processed, archival tumor tissues determined by three different anti-RB antibodies. International Journal of Cancer 58: 161-167.
•Skelly M, Coltrera MD, Gown AM 1996. Immunohistochemical analysis of p110RB expression in human cells and tissues. A reappraisal and critical review of the literature. Applied Immunohistochemistry 4: 16-24.
•Xu HJ, Cagle PT, Moore GE, Benedict WF 1991. Absence of retinoblastoma protein expression in primary non-small cell lung carcinomas. Cancer Research 52: 2735-2739.
•Xu HJ, Cairns P, Hu SX et al 1993. Loss of RB protein expression in primary bladder cancer correlates with loss of heterozygosity at the RB locus and tumor progression. International Journal of Cancer 53: 781-784.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
Accurate (84B3-1), Biodesign (RB1, 1F8), Biogenesis (RB), Biomol Research (MAB245), Dako (Rb1), Labvision Corp (1F8), Novocastra (Rb1), Oncogene (AF11, LM95.1), Pharmingen (245), QED (3C8) and Santa Cruz (C-15)
Fixation/Preparation
The antibodies are mostly immunoreactive only in fresh-frozen sections although some antibodies stain fixed paraffin-embedded sections but only after HIER.
Background
The Rb gene is located on chromosome 13q14 and spans a region of more than 200 kb, including 27 exons. The Rb gene is the only tumor suppressor that has been shown to directly suppress tumor formation. The Rb protein has a molecular mass of 105 kD and a number of antibodies which recognize specific parts of this protein have been developed. Besides loss of function due to chromosomal abnormalities including chromosomal deletion, translocation and point mutation, as with p53, phosphorylation may inactivate the Rb protein. In addition, a variety of viral oncoproteins including simian virus 40 T antigen, E1A from adenovirus and E6 from human papilloma virus may bind and inactivate the Rb protein. Immunostaining may be a valid way to assess the presence of normal Rb protein but several factors affecting staining should be considered before accepting the relevance of the technique. Firstly, it has been observed that the level of expression of Rb protein is not the same in all cells in any individual tissue, e.g. in the epithelium of the cervix, there are low or undetectable levels of staining in the basal layers and staining increases with cell maturation. In contrast, low or absent anti-Rb protein staining was observed in the well-differentiated epithelial cells of the gastric mucosa such as the foveolar and mucus cells compared to the cells in the crypts and neck of the glands. Astrocytes and microglia do not show detectable Rb protein by immunostaining and other subsets of normal cells such as some stromal cells do not display demonstrable Rb protein. The reasons for failure to demonstrate the protein at an equivalent level in all cells may relate to variations in expression as a function of cell cycling activity, cell differentiation and protein phosphorylation. More importantly, there is a large subset of cells, which RB include endothelial cells, lymphocytes and stromal cells, in which the ability to demonstrate p110expression is critically dependent on the method of staining used (Cordon-Cardo & Richon, 1994).
Applications
The p53 and retinoblastoma (Rb) gene products must be the two most-studied tumor suppressor genes. While alterations in the p53 tumor suppressor gene have been recognized as the most frequent genetic alterations in human neoplasia, the extent of Rb gene alterations is less well known. p53 alterations are mostly detected as overexpression of the protein and can easily be done with immunostaining, whereas most normal cells do not contain stainable wild-type p53 protein. In contrast, the Rb protein is detectable immunohistochemically in normal non-transformed cells, although whether this is so for all normal cells and tissues is currently unknown. As abnormality is based on the absence of stainable Rb protein, it is critical that techniques of maximal sensitivity must be employed and internal positive controls must be present in the sections (Skelly et al, 1996).
Alterations in the RB gene have been described in a number of human tumors including retinoblastoma, osteosarcoma, other sarcomas, leukemias, lymphomas and certain carcinomas including those from the breast, prostate, lung, bladder, kidney and testis (Geradis et al, 1994). Rb gene alterations have been associated with increasing tumor grade and stage in a variety of tumors and there is increasing evidence that alterations of this gene are associated with increased risk for metastasis (Xu et al, 1991, 1993). In breast carcinoma there is some evidence of association with other signs of progression and loss of hormonal receptor expression (Drobnak et al, 1993).
Comments
It was recently demonstrated that HIER in citrate buffer at pH 6.0 with overnight antibody incubation produced maximal sensitivity when staining fixed paraffin-embedded sections. Fixation in methacarn also requires HIER treatment and the use of DNAse produced variable results. The use of low pH buffers can produce false-positive results. Thus, in the assessment of Rb protein, as with other fixation-sensitive antigens, it is clear that the findings of individual laboratories cannot be generalized owing to differences in fixation and immunolabeling techniques. However, these factors do not preclude the assessment of the Rb protein in laboratories where fixation and other variables are strictly controlled.
References
•Cordon-Cardo C, Richon VM 1994. Expression of the retinoblastoma protein is regulated in normal human tissues. American Journal of Pathology 144: 500-510.
•Drobnak M, Cote RJ, Saad AD et al 1993. P53 and Rb alterations in primary breast carcinoma: correlation with hormone receptor expression and lymph node metastases. International Journal of Oncology 2: 173-178.
•Geradis J, Hu SX, Lincoln CE et al 1994. Aberrant RB gene expression in routinely processed, archival tumor tissues determined by three different anti-RB antibodies. International Journal of Cancer 58: 161-167.
•Skelly M, Coltrera MD, Gown AM 1996. Immunohistochemical analysis of p110RB expression in human cells and tissues. A reappraisal and critical review of the literature. Applied Immunohistochemistry 4: 16-24.
•Xu HJ, Cagle PT, Moore GE, Benedict WF 1991. Absence of retinoblastoma protein expression in primary non-small cell lung carcinomas. Cancer Research 52: 2735-2739.
•Xu HJ, Cairns P, Hu SX et al 1993. Loss of RB protein expression in primary bladder cancer correlates with loss of heterozygosity at the RB locus and tumor progression. International Journal of Cancer 53: 781-784.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
