Hii...it's quite possible..you should use milli q water for all preparations including washing buffer (PBST). You should also consider the pH factor in all aspects: antigen retrieval, washing buffer, Blocking solution. I think it will work.

Gud luck..

Thanks, Meetu and Moritz!

The pH of all the solutions (except for this Double-distilled water) were measured before being used and were ok. We didn't contemplate water pH. 

Tomorrow we will make it again, but with distilled water (as the adjusted protocol says). Hope it works and lets us continue.

Thanks again!

Regards,

Juan

Hii..why don't you go for milli q water?? There is no specific protocol of double distilled water. Maintain proper pH, temp like storage temp as well as incubation temp., concentration, incubation time and use miili q water, you will get the results surely.

Regards..

Some weeks ago an Uruguayan Doctor went to display and adjust the protocol. They used distilled water and the results were good. That's why tomorrow the IHC will be done with distilled water. 

I really don't know if Mili-Q water is available at the University. I'll tell what you say to my sister.

Thank you!

Hi

We worked with distilled water,deionised water or double distilled water and milli Q water. As long as PH is ok it does not effect the results which is negative. Could you try to  increase the incubation time either in room temperature or 4 degree which may cause the negativity of the results and could try with different concentration of the antibodies in the same environment. Hope this help.

From experience it highly unlikely that any kind of water could affect the state of your staining, however, as it was stated by the above scientist, the pH has to the culprit. Make sure that your antibodies (both primary and secondary were kept in adequat temperature as suggested by the manufactures', and last but not least the proper controls, both negative and if possible positive.

good luck

As I understood, the IHC kit you've used had never shown positive result? Can it be due to problems with AB? Of course it can! We're waiting for your results with distiled water, and if they fail, I advice you to contact with manufacturer due to AB kit defect.

Water can affect IHC staining. But since it is presumably being used with buffered saline (Tris or phosphate buffers), it should not present a problem, suggesting that the problem is elsewhere (antibody, fixation, tissue preparation). The only problem we've encountered with water is when it wasn't distilled or purified in some manner. 

We have used milli q water or distilled water, you could check with other steps and incubation timing. Did the supplier give you any positive control slides. Its always useful to use both positive and negative controls in your experiment

In which steps do you use (double) destilled water? Only for the preparation of the buffers? In that case I don't think, it will affect your results very much. For rinsing during staining steps sometimes simple tap water instead of destilled water is used (due to osmotic effects mainly). Can you give a more detailed protocol of your procedure? That migh help to get an idea, where your problems might be. As Tahmina already said: Positive (and negative) controls are essential! Does the doctor use exactely the same antibody (same supplier, same order number, maybe even same lot) in the same dilution?

Hello you all!

Finally, it worked, twice already.

Thank you all very much! You were helpful and informative, since I'm learning.

Xylene, PBS mother solution and Na Citrate were changed. The kit and antibodies are always the same. We put attention to pH, tried to maintain a higher room temperature (we are on a very cold spring right now), and put positive and negative controls. So, it worked and finally we could start with the definitive sections.

I attach protocol table I did (to check while we go forward), if anyone want to see it (It's in Spanish, sorry about that).

Regards!

(And sorry for any English mistake)

Wonderful, good to know the details.