Abcam Ad

Author Topic: Washing methods  (Read 2663 times)

0 Members and 1 Guest are viewing this topic.

Offline ASluo

  • GoldMember
  • *****
  • Posts: 121
Washing methods
« on: April 12, 2008, 02:39:27 AM »
HI guys, I've been having problems with normal frozen mouse spleen and lymph node sections coming off the slides after the staining procedure. The parts which remain, the morphology is very poor. I've asked around and the general consensus is that it might have been caused by water in the acetone, an adhesive (Starfrost slides) and/or  technical issues. At the moment I wash my slides very gently with 1X TBS (no detergent added) using a squeeze bottle. I was advised to use a rack and dip it in TBS or use a shaker because the flowing effect is way too harsh on the very fragile frozen sections. However, when I used mouse colon I didn't experience such problem with very good morphology. It could be a tissue specific thing? What are your thoughts??

Many thanks,
Annie

Washing methods
« on: April 12, 2008, 02:39:27 AM »

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
Re: Washing methods
« Reply #1 on: April 12, 2008, 08:14:21 AM »
How long have the sections dried? Longer drying time should help before and after acetone fix.

Have you used H2O2 blocking? Be careful the concentration and dilution agent. If you do, at what step did you do this? Before or after antibody incubation? This will affect the results too.

If you can provide detailed procedure, It may be helpful for us to solve the issues.

 

Offline ASluo

  • GoldMember
  • *****
  • Posts: 121
Re: Washing methods
« Reply #2 on: April 13, 2008, 08:27:34 PM »
The tissues were sectioned by our histology department and I believe they let them dry for 30 minutes after acetone fixation. I do not use hydrogen peroxide blocking however, one of my colleague working on the same slides before me used HCl (at 0.1 M) to block the endogenous alkaline phosphatase because of the crazy background. It successfully removed the background but it was also abolishing most of the antigen epitopes (especially foxp3, CD19 and CD4) so since then I've removed it from the protocol. 

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
Re: Washing methods
« Reply #3 on: April 13, 2008, 08:43:10 PM »
My suggestion is to use fresh cold acetone to fix sections and air dry longer than 30 minutes before and after.

Offline ASluo

  • GoldMember
  • *****
  • Posts: 121
Re: Washing methods
« Reply #4 on: April 13, 2008, 08:50:04 PM »
I believe they used dry acetone but water might have got into it and caused the poor morphology?

Re: Washing methods
« Reply #4 on: April 13, 2008, 08:50:04 PM »