Hello, so I am doing an experiment where I am trying to locate specific transcription factors in the mouse brain. I have decided to use a dual probe fluorescent in situ hybridization kit that I ordered from a company. However, the protocol that the company provided starts at the hybridization step, but I need to know how to permeabilize the tissue. I am doing the procedure as free floating tissue at 30um thickness and was wondering if anyone had any ideas on how to permeabilize the tissue? So far I have not received any specific fluorescence and I believe it is because I am not permeabilizing the tissue enough.
I regularly use .1-.2% TritonX 100 to permeabilize 40um mouse brains. This should work--- what are your currently doing to permeabilize the brain?
Brian Telfer
mentions above the possible use of saponin, and though I agree this is a great permeabilizing agent, it is less harsh than TritonX and I reserve its use for monolayer cell culture immunochemistry (at a concentration of .1%). Good luck!
We usually use 0.3% triton X-100 plus 5% bovine serum albumin (BSA) diluted in 1x PBS then filteration. We use it for both permeabilize and blocking of free floating mice brain section for 1 hour at room temperature. @
Permeabilization should not be a problem here, even if you use long probes. When I first started to set up a FISH protocol, I had no permeabilization step and it worked anyway, on 30µm free-floating mouse brain sections. Now I'm just doing the first washes with PBS+0,1% Tween20 to provide some permeabilization, but all the answers that have been given before are perfectly correct.
However, I've noticed that it works much better with cryostat sections stored at -20°C in an anti-freezing solution rather than vibratome sections.
Also, you might need to add some pre-treatment steps apart from permeabilization. For example, if you use RNA probes, you really need to do an acetylation step (0,25% acetic anhydride in Triethanolamine for 10min @RT). Some people also use proteinase K to break the cross-links induced by formaldehyde fixation that can prevent the probe to access the target (in my opinion, it is not necessary). In any case, you should ask the company from which you bought the kit, as they must know which pre-treatments can help. Good luck!
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence