Double in situ hybridisation protocols - UCL

1 In situ hybridization protocols By Pringle and W. D. Richardson Wolfson Institute for Biomedical Research and Biology Department University College London Gower Street London WC1E 6BT Tel 02076796724 or 02076796736 e-mail In situ hybridization showing labelling of individual cells expressing Fibroblast growth factor receptor 3 (Fgfr3) in an adult mouse brain coronal section developed with NBT/BCIP. Table of Contents OVERVIEW OF POTENTIAL 3 1. 3 2. TISSUE 4 3. HYBRIDIZATION 4 4. CORRECT PH .. 4 MINIMAL 5 IN VITRO TRANSCRIPTION OF LABELED 6 DIG labeling mixture for in 7 TISSUE 9 CRYOSECTIONING.

2 11 KNIFE 11 CUTTING FROZEN 12 WRINKLE-FREE FROZEN IN SITU DETECTION METHODS, CHROMOGENIC AND 14 1. NBT/BCIP. NITROBLUE TETRAZOLIUM SALT + 1. 14 2. INT/BCIP P-IODONITROTETRAZOLIUM VIOLET + NITROBLUE TETRAZOLIUM 14 3. FAST 14 4. FLUORESCENT 14 IMMUNOHISTOCHEMISTRY AFTER IN SITU 15 SINGLE AND Double IN SITU HYBRIDIZATION WITH DIG AND/OR FITC-LABELLED 16 Hybridization 16 10x "salts".. 17 How to deionize 17 Making RNase-free tRNA by phenol/chloroform 17 POST-HYBRIDIZATION 18 18 BLOCKING OF 18 Blocking 19 ANTIBODY 19 FIRST COLOUR 19 POST-ANTIBODY WASHES AND COLOUR 19 Pre-Developing 20 Developing 20 NBT/BCIP 20 INT/BCIP 21 10% (w/v) polyvinyl alcohol stock solution (PVA).

3 21 KILLING THE FIRST AP 21 SECOND COLOUR 22 FLUORESCENT IN SITU HYBRIDIZATION USING TYRAMIDE SIGNAL AMPLIFICATION (TSA).. 23 TSA Amplification 23 Killing 24 FAST 25 IMPROVED SENSITIVITY 26 IN SITU HYBRIDIZATION ON CELL 27 2We have used in situ hybridization in our laboratory since the early days of S35 labelled probes (now superseded) and have extensive experience of the various methodologies used and the many potential problems! This document contains detailed protocols for both single and Double in situ hybridizations and lists various methods of visualizing your signal, both chromogenic and fluorescent.

4 In situ hybridization is a multi-step process with many potential pitfalls, which can have a cumulative effect on the sensitivity of detection. Over the last ten years or so we have encountered many of them - usually by trying to take shortcuts! The lesson is: don t take shortcuts, no matter how trivial they might seem. For example, you can sometimes substitute formamide straight from the bottle for de-ionized formamide in the hybridization mixture, without much effect on sensitivity. Problems start when you purchase a bad batch of formamide (formamide degrades to formic acid), your in situs stop working and it takes weeks of trouble-shooting to get back on track.

5 Better to have stuck to de-ionized formamide in the first place. It is rarely a single problem that causes poor in situs, usually several things combine to frustrate you. This makes it all the more important not to change things unnecessarily. With a complicated protocol if it ain t broke, don t fix it. Overview of potential problems There are at least four major areas to pay really careful attention to: 1. Probe Choice of cDNA probe can be very important - size can and does matter. We prefer larger (approx 1 kb probes) although we do use smaller ones and larger ones. Small probes usually give less signal-to-noise. The level of expression of your target mRNA can become the limiting factor here.

6 We have found that cleaning our cDNA templates with phenol/chloroform and re-precipitating before in vitro transcription routinely generates good probes. Problems in this area have been traced to dirty templates. You can sometime get away with not cleaning your linearized cDNA, but this is not recommended. Transcribing the RNA probe correctly is paramount. If your probe doesn t look like the examples shown in this protocol , start again. You should have a nice tight band, not a smear on the gel. After transcribing our RNA probe we do not remove the cDNA template nor the free nucleotides. Experience has taught us that there is no advantage in doing these extra steps and in fact we have found they can decrease the sensitivity, perhaps because of loss of total probe.

7 If you use the quantities outlined in the transcription protocols you will find it unnecessary to quantify the amount of probe generated. Invariably they work at a 1/000 dilution. Sometimes we lower the concentration. Higher concentrations ( 1/500) tend to give a high background. 32. Tissue Preparation We routinely fix in 4% paraformaldehyde and cryoprotect in 20% sucrose solutions which have been Diethyl Pyrocarbonate (DEPC)-treated, to destroy any residual RNase. Fixation times are important; over- or under-fixation can reduce sensitivity, perhaps related to reduced probe penetration into the tissue (over-fixed) and degradation or loss of mRNA (under-fixation).

8 Preparation of the sucrose solution is paramount. We always DEPC treat the solution and then autoclave. Dissolving sucrose in RNase free water might work some of the time, but leaves you vulnerable to an RNase contaminated sucrose solution, which is not a good idea. Problems have arisen in the past with non-DEPC treated sucrose solutions which have contained RNase and destroyed the mRNA in the tissue whilst cryoprotecting. Note that you cannot destroy RNase merely by autoclaving! 3. Hybridization buffer You must use de-ionized formamide routinely. Sorry, but that ultra pure Analar grade is not de-ionized formamide. (Note this doesn t matter for the high temperature washes after you have hybridized your probe, just in the hybridization buffer).

9 You must also take care to avoid any RNase contamination of the hybridization buffer. For example, you must ensure that the tRNA you use is RNase free (see protocol below). Buying molecular biology grade tRNA is no guarantee that it is RNase free. If it doesn t say it is RNase free on the label then it isn t. All other solutions should be bought in as RNase-free and solutions should routinely be DEPC. 4. Correct pH Use a Tris pH electrode. If your pH electrode is not specifically designed for Tris buffers your final pH will be incorrect (usually ~1pH unit out). Tris electrodes are made of glass, not the robust plastic ones seen in general use in most laboratories.

10 Those are fine for most reagents and buffers, but not for Tris buffers. If you haven t got one use pH paper (do not dip the paper in the buffer but take a drop of buffer to the paper using a sterile pipet!). The pH optimum for calf alkaline phosphatase (AP, the enzyme that ultimately develops your colour reaction) is so it will help if your developing solution is at Of course alkaline phosphatase will work at anything above pH8, so solutions that are not at the correct pH will ultimately yield a colour reaction, but not as efficiently and sensitively as one at 4 Minimal protocol Make DIG or FITC labelled antisense probes.