Taminem2012

I think you will find that it isn't so much about how the ions affect the cells, it will be more about how the ions affect the proteins they interact with.  In the case you described above, the triton (detergent) acts as a surfactant and helps puncture the membranes so that the rest of the solution can act more effectively as a hypo-osmotic solution, swelling the cell, and making the tight junctions more visible. The K, Mg and Ca are likely to be co-factors in the tight junction formation and/or permeability ( as a quick google search just confirmed).

There are plenty of reasons and some of them are;

• Mycoplasma contamination
  
• Prolong trypsinization
  
• Altered culturing conditions
  
• Improper cell-freezing

and many others


Cheers!

I have never heard of antibody staining done without some kind of coverslip (parafilm, in your case). I don't know why the first method wouldn't work.

Are your samples on a slide or on the petri dish itself? For samples grown on coverslips, I cut parafilm to fit inside a petri dish. Pipette antibody solution onto the parafilm (it will bead up) and gently place inverted coverslip w/sample onto the the solution (so sample is face down with a layer of solution between parafilm and coverslip. Petri dish was place in a humidity chamber (sealed plasticware with saturated paper towels/kimwipes).

I have also done this with samples on slides, adding solution to the slide and placing a coverslip on top of it. M-series Lifterslips work better that regular coverslips -- they allow for more solution and more movement of molecules/antibody in the solution.

Hi,
  I have a question regarding how primary and secondary antibodies should be incubated. I have tried two methods so far and have gotten drastically different results and wonder why this is so.

At first, I tried simply diluting the primary antibodies in 1%BSA and then adding it to the samples in a petri dish. On the sides of the petri dish are wet kimwipe so that when I close the lid of the petri dish, the primary antibody solution does not get dried. I incubated the secondary antibody the same way. After mounting, and looking at the pictures, my signal was very weak and I could see very few stained tight junctions proteins (my target protein).

On my second attempt, I decided to add parafilm on top of the primary antibody solution to spread out and flatten the solution on top of the sample. After mounting this time, the signal was a lot stronger and a lot more stained tight junctions can be seen.

1) Does anyone know why the second method gave me better results? 2) Is there any better method for incubating antibodies that you guys know?

Thanks.

Hi,
  I have questions regarding the pre-extraction step and several other steps of this protocol for epithelial cells' tight junction immunostaining.

http://www.invitroge...-Protocols.html

1) The protocol states that omission of this step causes no staining of tight junction but does anyone know the reason why it does that?
2) The pre-extraction solution is Trition-X diluted in Hepes Buffered Saline solution, is there a reason why it is diluted in HBS and not PBS?
3) Is BLOTTO analagous to BSA - bovine serum albumin?
4) When using mounting medium, ex. vectashield w/ DAPI, is there a difference between hard set and soft set?
5) For the sample preparation, why did the author use 50% confluency rather than 100% confluency prior to immunostaining?

I apologize for the many questions that I have, but if you guys can help me with any of these questions, I am more than grateful for your time and help. Thank you.