Loss of tissue mainly comes from inappropriate slide pre-treatment before the IHC.

Also, loss of  5% of the cores could be in the normal range.

Retrievals are the same as for full section IHC/Biopsy.

What is the core diameter that you have on the TMA? The higher the diameter-the lower losses you will have.

Cheers,

Ivan

I have used APES ((3-aminopropyl)triethoxysilane) coated slides for TMAs  (small and large cores). Before coating - a crucial step is a very thorough cleaning of slides with acid alcohol (1%HCl in IMS on orbital shaker for 1 h, then washing in 3 changes of dist H2O). Sigma Aldrich has a good protocol for slide coating on APES spec sheet. It's experimented with different AR for TMAs (tissue fixed in 4% paraformaldehyde) and what worked best for my antigens was citrate buffer pH 6.0 at 80degC for 1 h; slides in glass container in a water-bath. This AR is gentle and does not lift edges of the cores. You could try it - it might work for your antigens. Good luck with your experiments.

Thanks to both of you. I have tried 1 hour Xylene dewaxing previously as a TMA-IHC-protocol-modification. I will however try the gentler 80degC gentler AR.

Unfortunately, I buy slides in commercially so the fixing and coating is beyond my control. 

I would like to try and link up with a lab that produces TMAs for this reason.

Best,

Jason

Dear Jason, The procedure of coating slides is quite simple, so you might file it for your future projects. If you have to rely on commercial sources - I hope you are using positively charged slides such as "X-tra(TM) Adhesive" from Leica Microsystems (in UK available from Surgipath). A box contains 72 slides - but  for TMAs you don't need many. The most important is not to lose tissue cores and not to cut too deep into the block (some cores are usually a bit shorter than others and you would have empty spaces  instead of tissue) . If your lab doesn't use positive charged slides it is worth buying them or asking another lab at the University if they could give you some. If you can't get any - I could send you a box (you would need to send me your address).

You can use adesive tapes to reduce core losses, but it will cost you more.

We used some alternative silanization methods, you can find it in our papers to downloud.

Since you do not produce the TMAs by yourself, a lot of conditions are beyond control.

You can try to build TMA arrayer on your own or to buy some of the available manual arraying kits.

Then you will have a bit more confidence in the arrays.

Cheers and good luck,

Ivan

Since your TMAs are commercial and you have trouble with them, you may consider looking for an alternative vendor. Some of the commercial TMAs have a lot of "bad" cores anyways and very variable quality within the TMA. Some cores are fine some may not even give you a decent signal for a marker such as Ki67. SInce these TMAs are generally pretty expensive I always do at least a double immunofluorescence and do one antigene that is my "normalizer" to decide which cores can be evaluated or compared to the other cores. In the end you may be better off making your own TMA or staining samples individually obtained from another source. The CHTN is a great source for human tissues (http://www.chtn.nci.nih.gov/).

To troubleshoot:

1-Bake your slides overnight at 56C. This would help to keep your slides

2- Citrate buffer has to go above 95 but under 100C to reverse the crosslinking and unmask your antigens. Never make it BOIL.