Question (25946) | Goat Anti-Rat IgG H&L (Cy5 ®) preadsorbed (ab6565)

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Question

I send in attach the protocol I used (that unswer to all questions of the inquiry). This protocol is optimized for a lot of antigens, namely CD31....   Fixative for 10 min at RT: PFA 4% Acetone PFA:Acetone (1:1) Wash 3x in PBS Permeabilize with Triton X-100 0,2%, 10 min, RT; Wash 3x in PBS Blockinh with BSA/PBS 1%, 40-60 min, RT; Wash in PBS Incubate 1st antibody, ON, 4ºC (rat CD31 abcam, ab7388) 1:50 1:100 1:500 1:1000 Wash in PBS Incubate with 2nd antibody in the dark, for 2h, RT (anti-rat abcam, ab6565) 1:1000 1:500 Wash in the dark Coverslip with Prolong Gold + DAPI. Fixative for 10 min at RT: PFA 4% Acetone PFA:Acetone (1:1) Wash 3x in PBS Permeabilize with Triton X-100 0,2%, 10 min, RT; Wash 3x in PBS Blockinh with BSA/PBS 1%, 40-60 min, RT; Wash in PBS Incubate 1st antibody, ON, 4ºC (rat CD31 abcam, ab7388) 1:50 1:100 1:500 1:1000 Wash in PBS Incubate with 2nd antibody in the dark, for 2h, RT (anti-rat abcam, ab6565) 1:1000 1:500 Wash in the dark Coverslip with Prolong Gold + DAPI.  

Answer

Thank you for your response. Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem. 1)  Sample: Could you specify the samples used for the staining i.e. species, cells (cultured) or tissues? 2) Detection system: Have you used the secondary antibody successfully with another primary antibody? Do you think that the problem may come from the secondary antibody? Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

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