I have questions regarding the pre-extraction step and several other steps of this protocol for epithelial cells' tight junction immunostaining.
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.
Edited by Taminem2012, 27 July 2011 - 12:03 AM.