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Immunostaining Protocol Help


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#1 Taminem2012

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Posted 27 July 2011 - 12:00 AM

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.

Edited by Taminem2012, 27 July 2011 - 12:03 AM.


#2 bob1

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Posted 28 July 2011 - 02:18 AM

It is a guess, but the pre-extraction appears to be a permeabilisation step, which will poke holes in the membrane allowing antibodies to enter. Tradionally these are done after fixation; I don't know why they do it before fixing here as they have another afterwards, unless they are trying to partially separate the cells so that the tight junctions are exposed.

They use HBSS as it is a bit gentler on the cells. PBS should work too.

BLOTTO is "Bovine Lacto Transfer Technique Optimizer", it is analagous to BSA.

Hard set is a mounting medium that sets, soft doesn't

Cells at 100% tend to be hard to look at under the microscope as they are all tightly packed together packing the cellular components together making visualisation hard. 50-70% should allow the cytoplasm to spread out but still give tight junctions.

#3 Taminem2012

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Posted 28 July 2011 - 12:11 PM

Thank you for your help, bob1.

It is a guess, but the pre-extraction appears to be a permeabilisation step, which will poke holes in the membrane allowing antibodies to enter. Tradionally these are done after fixation; I don't know why they do it before fixing here as they have another afterwards, unless they are trying to partially separate the cells so that the tight junctions are exposed.

They use HBSS as it is a bit gentler on the cells. PBS should work too.

BLOTTO is "Bovine Lacto Transfer Technique Optimizer", it is analagous to BSA.

Hard set is a mounting medium that sets, soft doesn't

Cells at 100% tend to be hard to look at under the microscope as they are all tightly packed together packing the cellular components together making visualisation hard. 50-70% should allow the cytoplasm to spread out but still give tight junctions.






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