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Alpha B Crystallin Antibody Staining Protocol for Immunohistochemistry
Description: Alpha B crystallin is a lens protein which has some homology with the small heat shock proteins. It is expressed in tissues such as skeletal muscle, cardiac muscle, smooth muscle, renal tubular epithelium, Schwann cells, glial cells, thyroid epithelium, colonic epithelium and stratified squamous epithelium. Alpha B crystallin is also reported to be found in ubiquitinated intermediate filament inclusion bodies, such as Lewy bodies (neurofilaments), Rosenthal fibres (glial filaments) and Mallory bodies (cytokeratins). Alpha B crystallin is rarely found in neurofibrillary tangles. The role of Alpha B crystallin in these inclusion bodies is unknown, however, it may function as an accessory protein for intermediate filament aggregation.
Primary Antibody
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Name: alpha B Crystallin Antibody |
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Clone: G2JF, Mouse Anti-Human |
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Supplier: Novocastra Labs |
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Catalog Number: NCL-ABCrys-512 |
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Dilution: 1:80 - 1:160 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed. |
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Incubation Time/Temp: 60 min/room temperature |
Antigen Retrieval
| Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102) |
| Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100) |
| Heat/Cool Temperature: 95-100 ºC/room temperature |
| Heat/Cool Time: 20 minutes/20 minutes |
Detection Methods
| Standard Method: ABC Method or LSAB Method |
| Enhanced Method: Polymeric Methods |
Chromogen Substrate
| Reagent: DAB |
| Incubation Time/Temperature: 1-3 minutes/room temperature |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Cytoplasmic |
| Images: Search image |
Additional Information:
| Tissue Type: Kidney |
| Fixation: Formalin-fixed paraffin sections, or acetone fixed frozen sections |
| Positive Control: Kidney |
| Negative Control: Omit primary antibody, isotype control, absorption control |
| Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary |
Notes:
There is no need to perform antigen retrieval on acetone fixed frozen sections |
References:
1. McQuaid S, McConnell R, McMahon J, Herron B.
(1995) Microwave antigen retrieval for immunocytochemistry on
formalin-fixed, paraffin-embedded post-mortem CNS tissue.
J Pathol. 176(2):207-16.
PubMed Abstract.
2. Cassiman D, Roskams T, van Pelt J, Libbrecht L, Aertsen P, Crabbe T, Vankelecom H, Denef C. (2001) Alpha B-crystallin expression in human and rat hepatic stellate cells. J Hepatol. 35(2):200-7. PubMed Abstract.
3. Ishiguro Y, Kato K, Akatsuka H, Iwata H, Nagaya M. (1997) Chemotherapy-induced expression of alpha B-crystallin in neuroblastoma. Med Pediatr Oncol. 29(1):11-5. PubMed Abstract.
4. Iwaki T, Iwaki A, Liem RK, Goldman JE. (1991) Expression of alpha B-crystallin in the developing rat kidney. Kidney Int. 40(1):52-6. PubMed Abstract.
5. Iwaki T, Wisniewski T, Iwaki A, Corbin E, Tomokane N, Tateishi J, Goldman JE. (1992) Accumulation of alpha B-crystallin in central nervous system glia and neurons in pathologic conditions. Am J Pathol. 140(2):345-56. PubMed Abstract.
6. Cooper PN, Jackson M, Lennox G, Lowe J, Mann DM. (1995) Tau, ubiquitin, and alpha B-crystallin immunohistochemistry define the principal causes of degenerative frontotemporal dementia. Arch Neurol. 1995 Oct;52(10):1011-5. PubMed Abstract.