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Dernière publication 01/01/2024

Up to the maximum-testosterone dose-dependent effects on anabolic and androgen r

The antigen retrieval for the prostate slides was carried out in 10mM Tris-EDTA (pH 9.0) at 60C over night. Then, the Millipore IHC SelectHRP/DAB...

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    [title] => Up to the maximum-testosterone dose-dependent effects on anabolic and androgen r
    [paragraph] => Up to the maximum-testosterone dose-dependent effects on anabolic and androgen responsive tissues in orchiectomized rats
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Authors
Daniel Derwand, Oliver Zierau, Detlef Thieme, Annekathrin Martina Keiler

 Lab

Journal
Andrology
Abstract
The antigen retrieval for the prostate slides was carried out in 10mM Tris-EDTA (pH 9.0) at 60C over night. Then, the Millipore IHC SelectHRP/DAB Kit was used for the nuclear staining with KI67-antibody according to manufacturer's specifications with the following modifications. Endogenous peroxidases were blocked with 3% H2O2for 10min. The blocking was carried out with 20% bovine serum albumin (BSA) in 1xTBS for 1h at room temperature (RT) followed by the primary antibody incubation for 1h RT (rabbit monoclonal Anti-Ki67 antibody [SP6] ab16667, abcam, Lot no. GR3313195, 1:100 in 20% BSA in 1x TBS) in accordance to Chen etal. (2013).22The biotinylated secondary antibody, streptavidin-peroxidase-complex, DAB-solution and hematoxylin solution were provided and used according to the Millipore IHC SelectHRP/DAB Kit. The ready to use secondary-antibody was incubated for 1h RT, the streptavidin-peroxidase complex for 10min, the DAB solution for 4min, and hematoxylin for 10min (followed by 10min bluing in hand warm tap water). As washing buffer were 1xTBS or H2O used according to the kit manual. The stained slides were dehydrated and covered as mentioned before. IgG isotype control was carried out under the same conditions as for the KI-67 Antibody with rabbit IgG-antibody (monoclonal, (EPR25A)-Isotype control, ab172730, abcam, Lot No. 1011223to4, 1:100 in 20% BSA in 1x TBS). For the determination of KI67 positive nuclei, randomly chosen image sections (200x magnification) were completely counted (minimum 200 nuclei per animal). One technical replicate of each animal was evaluated. For the evaluation of the prostate epithelial thickness, three images (200x magnification) from five animals per treatment group were randomly chosen for measurement of epithelial thickness (10 per image).
Keywords/Topics
testosterone;dose-dependent;anabolic;androgen;responsive;tissues;orchiectomized;antigen;retrieval;prostate
BIOSEB Instruments Used:
Bioseb CIS Software (BIO-CIS2),Grip strength test (BIO-GS4),Rodent pincher - Analgesia meter - Wireless (BIO-RP-WRS)
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