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Thank you for your response, I will answer the questions 1. Samples: What sort of samples, species, tissue or cell type you used? brain sections (50um) from C57BL6 mice 2. At what dilution of this primary antibody you applied? What buffer you used for diluting this product? We tried different concentrations of the ab (1:1000, 1:500 and 1:100) in Tris-buffered saline (TBS: 50mM Tris + 0.9% NaCl pH 7.4) containing 0.5% (v/v) Triton X-100 and 1% of Horse serum) 3. Fixation: What percentage of PFA, for how long and at what temperature you used for fixation? 4% PFA at room temperature during 48h. 4. Permeabilization: At what percentage of Triton you used? For how long? all the solutions, washes, blocking serum and primary ab incubations are in Tris-buffered saline (TBS: 50mM Tris + 0.9% NaCl pH 7.4) containing 0.5% (v/v) Triton X-100. The blocking with horse serum is for 1h and the incubation with the primary is for 48 h. (everything at room temperature). 5. Have you applied any positive control along with the samples? No.
Asked on Aug 25 2006
Thank you for taking the time to fill in our technical questionaire and send it in to us. I am sorry you have had difficulty obtaining satisfactory results in immunohistochemistry with this antibody. I have read through the details and would like to make a few suggestions that I hope will help if you have not tried them already. 1. Your incubation time with primary antibody is very long. I would suggest 1 hour at a 1:100 dilution or 4oC overnight. 2. The target protein (TCR) is a membrane protein. Triton 100 is not suitable for membrane proteins, as it is a very strong detergent and can disrupt the membrane proteins and therefore the epitope for the antibody. I would suggest trying Tween 20 instead. There should be no requirement for permeabilisation to enable the antibody to access the epitope. However, on some rare occasions, antibody epitopes of membrane proteins are on the cytoplasmic side of the membrane (this will obviously depend on the antibody clone). If you obtain no staining after changing to Tween 20, you could try an acetone permeabilisation step. 3. Have you tried enzymatic antigen retrieval with for example proteinase K or trypsin? Again, this may be more gentle. 4. To the best of my knowledge, the C57BL6 mice should express TCR. There should be no difference between this and the TCR receptor expressed by BALB mice. 5. I would strongly recommend including a positive control. This could be lymph node or spleen samples from mice. I hope this is helpful. Please do not hesitate to get back in touch if you require more assistance.
Answered on Aug 25 2006