I guess in your case the purple precipitate is a result of AP (alkaline phosphatase) converting tetrazolium salt into insoluble formazan, and the reaction is sensitive to several factors.The most important ones influence the activity of AP enzyme like inactivation of AP by phosphates like PBS can occur, also pH, time of reaction, etc. You might need to test this to make sure that AP gives a robust signal in your hands.

DAKO GenPoint system works great. It uses tyramide amplification and DAB as a substrate, which is also better to counterstain.

There can be several reasons for it, e.g. the cross-linking PFA-fixative reduces the accessibility of the RNA to be detected by the in situ hybridization (ISH) probes. Or the other way round, the penetration of the probe is not good enough. In my experience, the use of immersion-fixed or perfusion-fixed tissue (with or without paraffin embedding) for ISHresults in lower signal intensities compared to fresh-frozen tissue, cut at the cryostat, an post-fixed for a few minutes e.g. 4% PFA in PBS.

I guess in your case the purple precipitate is a result of AP (alkaline phosphatase) converting tetrazolium salt into insoluble formazan, and the reaction is sensitive to several factors.The most important ones influence the activity of AP enzyme like inactivation of AP by phosphates like PBS can occur, also pH, time of reaction, etc. You might need to test this to make sure that AP gives a robust signal in your hands.

Dear Dr. Nigam,

I think we need more information if we are to help you as best we can.

Specifically, are you using DNA oligos or riboprobes? How many bps in length are your probe(s). Are you using digoxigenin (DIG)-labeled dTPs? Are oligos being tailed or are you creating riboprobes via in vitro transcription? What is the composition of your hybridization solution/buffer? Have your already determined the optimal parameters for in situ hybridization (ISH) using your probe and target mRNA of interest (e.g., probe concentration, hybridizations temperature, stringency wash temperature etc.). Has your probe work well in the past and now not giving good signal, or is this the first time you have use the probe and you just notice a faint purple color where the mRNA should be in your tissue? Please give us some more background with regards to this information?

Without know the answers to the above questions, I am going to assume you are using DIG-labeled probes and an alkaline phosphatase (AP) conjugated antibody used to detect the DIG-label and cause the purple precipitate.

1. I agree, so does the field of scientists using in situ hybridization (ISH), with Dr. Schmitt that using fresh- and flash-frozen tissue, not perfusion-fixed tissue gives you the best ISH signal. I would also add that post-fixation (e.g., flash-frozen slide dipped in 4% PFA in 0.0M PBS pH 8.0) time needs to be empirically derived for your specific mRNA of interest.

2. In my experience (this is also in the literature in an empirically derived manuscript), you do not need to permabalized the tissue by using something like Proteniase K. All Proteniase K seems to do is degrade your histology and reduce your ISH signal.

3. Are you acetylating your tissue by using triethanolamine and acetic anhydride? Thins is an important step for all ISH and can greatly increase your signal if you are not doing it.

3. I agree with Dr. Wagner that the correct tyramide amplification (TSA Kit) can enhance a really low signal/rare transcript if your ISH condition are more or less already optimal (i.e., your ISH signal without the TSA kit is not going to get any better).

4. My last point is just to tell you what I use and works well for me in order to detect my DIG-label after my stringency washes and develop the purple precipitate in my tissue.

Anti-DIG-AP Fabs Roche# 11093274910 (1:2000 left on the tissue over night at 4 deg C)

NBT (nitro-blue-toluidine) Roche# 1383213

BCIP (Bromo-Chloro-Indoyl-Phosphate) Roche# 1383221

1X NTMT (50 ml)

5 M NaCl 1 ml

1 M Tris pH 9.5 5 ml

1 M MgCl2 2.5 ml

Tween-20 50 µl

Ultra Pure Water 41.45 ml

Developer

1X NTMT 1 ml

BCIP 3.5 µl

NBT 4.5 µl

I usually let the developer stay on the tissue for 1-2 hours after thoroughly washing off the Anti-DIG-AP Fabs. Development time needs to be empirically derived for your specific ISH conditions.

Using my protocol, I get good specific detection of both rare and abundant transcripts with a nice deep purple color.

I hope this helps.

Best,

Josh

Hi all,

I would like to thanks all of you for your valuable suggestions. I am mentioning below my complete protocol, which I used on my test as well positive samples taken on same slide.

dewaxing of 5micron paraffin sections.

Xylene washes.

Rehydration to PBS (alcohol gradation 100% to 25% ).

0.2N HCl treatment, 20 min at room tempt.

4% PFA fixation,10 min.

PBT Washes, 5min X3.

Proteinase K, 20min at room tempt (also tried 10 min in few slides).

PBT washes, 5 minX3.

4% PFA, 5min room tempt.

PBT washes.

Acetylation using triethanolamine and acetic anhydride, 10 min (also tried 20 min).

Air drying completely.

Probe (1microlitre probe in 100 microlitre Hybe solution, heated to 80 degree).

Hybridisation at 65 degrees overnight.

TNE washes(at 37degree, 10min) followed by SSC washes (65 degree, 20min).

MABT washes 5min at room tempt.

Blocking for 1 hour (using serum).

Anti-DIG-AP Fabs Roch (1:2500), at 4 degree overnight.

MABT washes.

NTM(pH 9.5)+NBT+BCIP, at room temperature till signal develops (in my case signal appeared after 10-12 hours).