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Our customer has tried the proteolytic pretreatment with 0.1% trypsin but the result again was not very convincing (see attached pdf-file 'PrlR-Staining'). The file shows pictures taken from stainings with AB2773 (page2) and pictures taken from stainings with the non-commercial antibody T6 (page1, from S. Kloehn et al.). The T6 antibody is described in: Ouhtit A, Ronsin B, Kelly PA, Morel G. Ultrastructural expression of prolactin receptor in rat liver. Biol Cell. 1994;82(2-3):169-76 Okamura H, Zachwieja J, Raguet S, Kelly PA. Characterization and applications of monoclonal antibodies to the prolactin receptor. Endocrinology. 1989 May;124(5):2499-508. So you can see that the results obtained with AB2773 are still not very good. If you have any more suggestions on how the results can be imroved they will be highly appreciated. (Please notice that I am on holiday next week, but my collegue Christian Erlenhoefer will take my place during that time) Previous message: I have a complaint about your Prolactin Receptor antibody #AB2773: Our customer used it in IHC staining on formalin-fixed, paraffin embedded tissue sections of rat liver and pituitary. Protocol: Antigen Retrieval: - with 10 mM Citrate buffer pH 6.0 using a Microwave Blocking: 5 min 0.5% Triton in TBS wash with TBS 20-25 min 0-35% H2O2 in TBS/Methanol (90/10) wash 3x with TBS 10 min DAKO blocking solution A 10 min DAKO blocking solution B wash 3x with TBS Primary antibody: AB2773 (Lot 19068) 1/50 in antibody diluent (DAKO) for 1h at RT wash 3x with TBS Secondary antibody: biotinylated secondary antibody from DAKO (LSAB-Kit, ready to use) for 30 min at RT wash 3x with TBS Streptavidin-Biotin-Complex (DAKO) for 30 min at RT wash 3x with TBS Detection with AEC-Substrate Kit. Counterstaining in Hämalaun, embedding in Kaisers Glycine-Gelatine. Problem: no specific staining could be obtained. The same protocol was used successfully using a Ki67 antibody on rat tonsil. Please hava a look at the attached pictures.
Asked on Jul 12 2004
Here is a reference that uses our monoclonal antibodies with IHC procedures: Rl Sorenson and LE Stout. Prolactin Receptors and JAK2 in Islets of Langerhans: An Immunohistochemical Analysis. Endocrinology 136:9 pp 4092-4098. 1995 Also used in FACS analysis: M. Gagnerault, et al. Expression of Prolactin Receptors in Murine Lymphoid Cells in Normal and Autoimmune Situations. J. Immunol., 150(12): 5673-5681, 1993. The customer should follow these protocols along with the other information have passed onto him/her with our previous correspondence. If not, please let us know how you would like to proceed. This will work, otherwise, there is another issue and not the antibody. Please let us know if this helps or if you require further assistance. Thank you,
Answered on Jul 14 2004