Ki67 IHC

[joshuaallen07]

I'm working with mouse brain that was transcardially perfused with 4% PFA, removed from skull, immersion fixed in 4% PFA for 48 hours, sunk in 30% sucrose, and sections on a freezing microtome at 40um.  Tissue is stained free floating.  I've worked with tissue processed this way for many years without problem, but I'm having a problem with my Ki67 IHC protocol.

When I mount the tissue and allow it to air dry, the tissue turns stark white and curls up.  Unless I wet the tissue with PB, the tissue will flake off the slide.  If I try to coverslip the tissue, it isn't flat and is folded.  I've never had a problem like this before. If I coverslip and look under the scope, the tissue looks awful - it looks like tiny microbubbles everywhere.  Any suggestions as to what is causing this?  I'll post the protocol below with some comments following.  Thanks!!

The protocol is as follows: 

Day 1:

1.   Remove tissue from cryoprotectant by washing 4 x 3 minutes in 0.15M phosphate buffer (PB)
2.   Incubate in L.A.B. solution for 20 minutes
3.   Incubate in 3% H2O2 for 20 minutes
4.   Wash 4 x 3 minutes in 0.15M PB
5.   Incubate sections in 10% normal goat serum (made in 0.15M Phosphate buffer + 0.4% Triton-X; PB-TX)   
        for 60 minutes
6.   Incubate in primary antibody (Vector anti-Ki67; 1:1500) in 0.15M PB-TX+ 1% NGS for 48 hours at 4*C

Day 3 (all steps at room temperature, unless otherwise noted):
1.   Wash 8 x 3 minutes in 0.15M PB
2.   Incubate sections in biotinylated goat anti-rabbit IgG (Vector; 1:2000) in 0.15M PB-TX + 1% NGS for 2     
hours
3.   Prepare ABC solution (Vectastain Elite Kit; Vector Labs PK6100)
a.   (2µL/mL Solution A + 2µL/mL Solution B) in a smaller volume of 0.15M PB then necessary
b.   Allow ABC solution to conjugate for 20 minutes & dilute to final volume with 0.15M PB
c.   Incubate in ABC solution for 1 hour
4.   Wash 4 x 3 minutes in 0.15M PB
5.   Wash 4 x 3 minutes in distilled water
6.   Prepare DAB solution ( according to manufacturer’s instructions) & incubate in DAB solution for ____ minutes
7.   Rinse in distilled water 5 times immediate after removal from DAB solution
8.   Rinse 4 x 3 minutes in 0.15M PB
9.   Store sections in 0.15M PB at 4*C until mounted
10.   Mount 2x subbed slides or Superfrost Plus slides and air dry overnight
11.   Coverslip with cytoseal 60.

LAB solution refers to Liberate Antibody Binding Solution.  I typically don't use this solution, but the lab where I obtained the protocol said it is 100% necessary for this staining.  Here is the data sheet:  http://www.polysciences.com/SiteData/poly/Assets/DataSheets/630.pdf

Also, I don't allow the slides to air dry overnight.  They tissue is dry to the point that it is curling at the edges within 15 minutes of mounting it onto the slide. 

Any thoughts would be greatly appreciated!! 

[gula]

As your cytoseal is toluol-based, you can try to dehydrate your slides freefloating through graded ethanol untill xylen.  Mount and coverslip the wet slides.

I don't believe, that the LAB solution can cause your problem. There are so many washing steps in between.
Do you usually also work with sections of that thickness? How do the sections usually behave while drying?
Have your tried to use a water-soluble mountingmedium?

gula

[joshuaallen07]

As your cytoseal is toluol-based, you can try to dehydrate your slides freefloating through graded ethanol untill xylen.  Mount and coverslip the wet slides.

I don't believe, that the LAB solution can cause your problem. There are so many washing steps in between.
Do you usually also work with sections of that thickness? How do the sections usually behave while drying?
Have your tried to use a water-soluble mountingmedium?

gula

I work with 30-40um brain sections all the time and I've never had this problem.  My standard protocol is similar to what is listed above, but I typically don't use the L.A.B solution.  I've stained for GFAP (DAB) and IBA1 (fluorescence) using a different series from the exact same tissue and I don't have this problem. 

I'm doing to remove the cover slips and float the tissue off the slides to see if I can remount the tissue.  I might give the water-soluble mounting medium a whirl as well. 

Thanks for the quick reply! 

[gula]

That sounds like one of your usual reagens may be wrong in some way.  Perhaps changing of the buffer may help.
gula