[Histonet] Fern tissue loss during in situ hybridization

[Cordle, Angela R]

Dear Histonet members,

I’m having problems with tissue loss during in situ hybridization (ISH) with fern tissue. The fern in question is Ceratopteris richardii, and the tissue (tissue does not contain spores) has been fixed in FAA, dehydrated and embedded in Paraplast plus. Sections are 7 um, and mounted on Probe on Plus slides. I’m not an expert at this process, but I’ve done a fair amount of embedding, sectioning, and staining of various tissues (on Haupt’s coated slies), and I’ve had success with Arabidpsis ISH. I’ll outline the relevant and troubleshooting procedures that I’ve gone through here:

1. Varied the fixation times and methods. Nothing seems to make a difference in the retention of the tissue on the slides.

2. Tried a brand new box of Superfrost Plus slides (the ProbeOn Plus slides are kind-of old), but the superfrost slides actually seemed to provide worse tissue retention than the ProbeOn Plus slides. Arabidopsis tissue prepared in parallel was fine on both slides.

3. For mounting, I float tissue on 37°C DEPC H2O in a waterbath that is free from lotions, or any other substance that I am aware of that can cause problems with tissue adherence. I remove the slides with the newly adhered sections completely vertically. Again, Arabidopsis sections mounted in parallel do not fall off slides in subsequent procedures.

4. I’ve paid a (perhaps) compulsive amount of time making sure that there are no blebs or bubbles of water underneath sections after mounting. Additionally, slides are dried for 1-2 hours (vertically) before baking them at 48°C. I have found that baking the slides for 2 days, rather than 1, slightly increases the tissue retention for the fern, but not enough for quality in situ results.

5. Varied the time and temperature of the proteinase K treatment, but the tissue seems to be falling off the slides before this step anyway. It is gone after this step for sure, regardless of the time or temperature.

5. Taken a great amount of care to ensure that there is no residual tert butyl alcohol (we dehydrate through an ethanol series them into 100% TBA before transitioning to Paraplast) in the paraplast before embedding and sectioning. Arabidopsis tissue embedded in the same blocks has performed perfectly fine in ISH.


Okay. So, here are my specific questions:

Could it be that this fern tissue is not sufficiently negatively charged that the tissue will not adhere to any polyL lysine coated slide?

What the beep should I try next?

In advance, thank you all so much for taking the time to help me out with this frustrating problem!    --Angie