I'm trying to make some DIG-labelled probes work, and I am not sure where to start trying to optimize the protocol. I'm doing ISHES on e12,5 mouse embryos, fixed ON in PFA 4%. My issue is that I get decent staining in the midbrain but I don't seem to get good staining in the forebrain, as it seems the gene is less expressed there. (Actually, I have issues with most probes in that area).
I've tried longer washes after the hybridization step just because I thought that would reduce the background and I would be able to stain longer, but it washed my probe as well.
If anyone has experience optimizing a protocol to make probes work, where do you start? Hybridization temperature? Length of washes? Is there anything else that can be played with?
If you need the complete protocol, let me know and I'll add that info.
Hi,
You can play with all of the different step ...
The complet protocol may be usefull
Hi! Here's the complete protocol I just translated in case any of you can help. Thank you very much in advance!!!
a- I prepare my sections rapid freezing the tissue in 10% gelatin 10% sucrose – PBS.
b- After sectioning, I let air dry ON and start the ISH protocol on the next day – I never freeze the sections because I found that could deteriorate my ISH (true?)
c- Hybridization day:
1- I refix the slides 10’ in fresh 4% PFA
2- 3 x 10’ washes in PBS
3- 15’ Acetilation: 50mL: 50mL H2O + 670uL x2 TEA 50% + 176 uL HCl 6M + 126 uL Acetic acid 100%
4- 30’ permeabilization in 1% triton-PBS
5- 3x10 washes in PBS
6- pre hyb: 400uL hyb buffer for at least 4 hours
7- Hyb: ON hyb at 72ºC with a certain concentration of probe (this is what I vary the most). Probes are heated to 80ºC and rapid-cooled before putting them onto the slides. Slides are covered with a cover glass and left in a humid chamber ON.
d- Inmunohistochemistry:
1- 2 x 45’ 0.2X SSC at 72ºC
2- 1 x 5’ 0.2 SSC at Room temp
3- 3 x 5’ washes with B1 solution (0.1 MTris pH7,5;NaCl 0.15M; 0.1% triton X100)
4- Blocking for at least 4 hours with B1 + 10% HINGS
5- ON incubation at RT with B1+1%HINGS+1:3500 anti DIG-AP
e- Developing:
1- 3x10’ washes in B1 sltn
2- 2x10’ washes in B2 sltn (0.1M Tris pH9,5;0.1M NaCl;0.05M MgCl2)
3- Developing in B3 (10 mL B2 + 200uL NBT/BCIP – Roche)
f- I develop 6h to ON. When developing ON, some precipitation is formed onto the slides that makes the background quite noisy, but then again some probes are very weak in my area of interest (tuberal hyp).
g- My probes vary from 200 to 1000pb in average, and are all dig-labelled.
h- I have a variety of probes (around 20) that I have developed myself or are taken from various papers. Some probes work when I adjust the concentration (I adjust them doing dilutions of the original synthesis reaction), and some others never worked although they are well characterized in the papers they are taken from, or even the allen brain atlas.
i- My lab mates are not used to doing ISHes, as I am the only one who works with them right now.
Hyb sltn:
-50% redistilled formamide
-10% dextran sulphate
-tRNA 1mg/mL
-1mg/mL Denhart’s sltn
-0.3M NaCL
-0.01M Tris
-0.005M NaH2PO4
-0.005M Na2HPO4
-0.005M EDTA
First i would try different hybridization temperature (from 55 to 72) because 72°C is maybe to restrictive and try to wash at a higher temperature than your hybridization temperature
Ok, then I'll go through the temperature path next time.
Wouldn't higher temperature washes wash too much of the probe if I'm hybridizing at, let's say, 65 and wash at 70?
1. Did you use DEPC water to prepare the solutions which are used before hybridization?
2. Although i didn't have experience of cryo-section nor animal tissue, i have worked on plant tissues for years. According to my experiences, I will suggest you to perform ISH soon after the sections are dried, since the RNA in tissue may have decayed before you fix it with PFA. Attached please find a protocol which works well in my lab. A published result is also attached for your reference.
Dear Mei-Chu Chung,
Thank you very much for your help. I will take a look at the protocol to see what are the differences with mine and check if there are advantages I'm not counting with. To answer your questions: 1. All my first day solutions are DEPC treated. The material I use is PFA-fixed ON before the first day to get rid of any RNAse activity left in them. 2. The animal tissue is fixed ON in PFA before sectioning, so that shouldn't be a problem either. I am aware that RNA decaying may be an issue but the truth is that some probes work with this protocol and some don't.
Thanks again and I'll take a look at the protocols you sent me.
-Daniela
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