Entangling of freefloating tissues during staining

[skumarmadhu]

Dear friends
I am new to this field and i am working from this feb on this topic

I will perfuse the mice brain(7day old pups) ,
take out the brain, fix it 24h in PFA
transfer to 1XPBS leave it for overnight at 4
embedd them in 3% agarose and take 40 micron sections
i wash them in wash buffer containing 0.1%triton-x100 in 1xPBS (10min)
block with 3%BSA, 10%NGS, 0.1%Triton-X100 in PBS (1h)
Primary antibody and secondary antobody-same blocking buffer except NGS

My tissues are very fragile and by the time i put them in the secondary they will become entangled, folded and unable to mount, i tryed to fig out the problem but disappointed plz can anyone help me in this regard

Madhu

[haisond]

Hi Skumar..

You are in luck, I've worked out all the issues with freefloating section IHC. I routinely stain 40-60 microns brain slices so i have become somewhat of an expert at this..

here are some solutions to your problem:
1. Buy a used Ronco rotiserie oven from Ebay (30 to 40 bucks), you can disable the heating element quite easily.
2. Buy a used well plate sealer ( I got one (Packard microplate sealer) for $50 bucks from Ebay).
3. Buy some thermal plate sealer film.
4. Place your specimens in 24-well plate, incubate in 750 ul - 1,000 ul of antibody solution
and seal wells with using the film and plate sealer. Your samples are now sealed and can be rotated in a rotiserie manner allowing for complete penetration of your tissue specimen from all sides, the constant motion will prevent tissue folding during incubation.
5. I place the rotisserie in a fridge to incubate samples at 4 degrees.
6. For mounting you should be concern with compression of your specimen by the weight of the coverslip. Buy some laminating film from McMaster Carr (catalog number 2271k72) these are 50.6 micron thick with an adhesive backing to tape onto your slide. Once the film is taped onto your slide, cut out an area large enough to fit your specimen. Basically, you will end up with a well that is 50.6 micron thick to place your specimen into. Place specimen into the well, use a one bristle brush (Electron Microscopy Science) to gently straighten out any wrinkling or folding. Once the specimen is in place remove excess liquid by dabbing with a kimwipe, do not let your sample dry out completely. Quickly add your mounting solution and coverslip with a 0 to 1 coverslip (EMS). I have many more tricks, if anyone else have issues with working with brain tissues.

good luck,