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LOT NUMBER -- NOT SPECIFIED -- oRDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No staining SAMPLE mouse adrenal gland PRIMARY ANTIBODY ab24345 DETECTION METHOD IF POSITIVE AND NEGATIVE CONTROLS USED None used -- can you recommend a positive control? ANTIBODY STORAGE CONDITIONS 4 degrees FIXATION OF SAMPLE 4% PFA, 4 degrees, O/N ANTIGEN RETRIEVAL tried with no retrieval and with SDS PERMEABILIZATION STEP 3% BSA, 3% goat serum, 0.3% Triton X-100, 0.3M glycine in PBS BLOCKING CONDITIONS Same as permeabilization SECONDARY ANTIBODY Alexafluor goat anti-mouse IgG1 HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes dO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES Can you provide a recommended staining protocol for tissue immunofluorescence for this antibody? (eg fixative, blocking, etc)
Asked on Jan 05 2012
Thank you for contacting us. I am sorry to hear your experiencing problems with our antibody. Could you please let me know what type of sections you are using? As of now, fixed-frozen or paraffin-embedded sections have not been tested with this antibody. Only IHC with perfused-frozen sections (IC-FoFr) has been performed, with an antibody dilution of 1/300 to 1/1000. The optimal dilutions would need to be determined by each researcher. As positive control rat cerebellum could be used as the antibody was tested on such sections. This is the protocol that has been used with the rat sections for perfused frozen sections: The immunohistochemical analysis was performed on sagittal Vibratome sections of 7-d rat cerebellum. Rats were perfusion fixed with picric acid-paraformaldehyde-glutaraldehyde, and sections were stained with peroxidase-conjugated second antibody as described below. Tissue fixation and staining. Sprague-Dawley rats were anesthetized with sodium pentobarbital (6.5 mg/100 g body weight) and perfused through the left ventricle, first briefly with 0.1 M phosphate buffer (pH 7.4), followed by the picric acid-paraformaldehyde-glutaraldehyde fixative described by Somogyi and Takagi (Somogyi, P., and Takagi, H. (1982) Neuroscience 7, 1779-1783). The brain was removed and allowed to stand in fixative for 2h at room temparature before being placed at 4 C overnight. After washing several times with PBS, the cerebellum was sectioned with a Vibratome to 15-30 um thickness for light microscopy. All sections were washed with 50 mM TBS (pH 7.6) before antibody staining. Sections were washed for 30 min in TBS containing 0.5% H202, 3 x 15 min in TBS, followed by 1h in TBS containing 5% BSA, 1% normal rabbit serum, and 0.2% Triton X-100. They were then incubated overnight at 4C in TBS containing monoclonal antibody, 0.5% BSA, 0.1% normal rabbit serum, and 0.2% Triton X-100. After washing for 1 h at room temperature in several changes of TBS without Triton X-100, the sections were incubated for 1.5 h at room temperature in TBS containing 0.5% BSA, 0.1% normal rabbit serum, 0.2% Triton X-100, and peroxidase-conjugated rabbit anti-mouse immunoglobulins (1:200). The sections were then washed for 1 h in several changes of TBS without Triton X-100, followed by 12 min incubation with 0.05% 3,3'-diaminobenzidine/0.01% H202 and 3 x 10 min washes in TBS. Sections were mounted on gelatin-coated slides, dehydrated through a series of graded ethanols, cleared with xylene, and sealed in Permount. Please let me know if this protocol is helpful to you and gives the desired results. If not, please let me know as well. I wish you good luck and look forward to hear back from you.
Answered on Jan 05 2012