Background in immunofluorescence staining

Use this category for questions regarding various immunological methods

Moderator: mdfenko

Background in immunofluorescence staining

Postby SebZ » Jul 01 2016 3:35 pm

Dear users,

I'm not sure whether I can just post like this since I'm new, but since I'm struggling a bit with my immunofluorescence staining I thought I'ld give it a go. I know there are probably other people with similar problems, however since my experimental settings are a bit unusual I'm not sure whether other topics would be directly applicable to me.

The thing is I'm trying to stain cells on a microchip that is placed in a 6-well plate. The staining works but I'm having a bit of issues with the observed background (see images in the attachment). However, my staining on cells in 96-well plates (not containing a microchip layer) looks fine. Although, that is a slightly different protocol (as in less extensive washing). I think its not something thats caused by my protocol, since its basically the same but only has more extensive washing steps; which in theory should get rid off the background since I use detergent for my washes. However, my protocol might be something that would need an independent check-up. I have colleagues using immunofluorescence as well, but they are not using the microchip platform so for issues related to this I'm pretty much on my own. Since the protocol (at least the version with less extensive washing) is based on a publication I'm starting to think that perhaps the background might be something thats inherent to the microchip (made of polystyrene) or perhaps that its hard to image the chip on our microscope (Operetta platform from Perkin Elmer) since the light source has to travel trough a layer of glass (from the 6-well plate), then the polystyrene and then the cover slip. The latter especially since we have a correction collar on the microscope that requires manual adjustment.

The protocol that I use is the following:
- Fixation with 4% PFA for 10-15 mins at room temperature (RT)
- Rinse 3x PBS
- Permeabilisation with 0,2% Triton X-100 for 10 mins
- Rinse 3x PBS
- Incubate in blocking buffer (PBS + 10% FBS & 0,25% cold water fish skin gelatin) for 1h at RT
- Wash 3x10 mins PBS (put plate on rocking platform)
- Label with primary Abs (2 microgram/ml) overnight at +4 degrees.
- Wash 3x30 mins with PBS + 0,2% Tween20 (plate on rocking platform)
- Label with secondary Abs, DAPI and phalloidin (ActinRed) for 1h at RT
- Wash 3x30 mins with PBS + 0,2% Tween20 (plate on rocking platform)

Any suggestions are welcome. :)

Image
Image
Image
Image
SebZ
newcomer
newcomer
 
Posts: 1
Joined: Jul 01 2016 2:32 pm

Return to Immunology and Immunochemistry Methods

Who is online

Users browsing this forum: No registered users and 1 guest