in situ hybridization not working - (Jul/05/2006 )

HELP! I've been working on in situ for months now, with no success. I'm using paraffin-embedded leaf tissue with DIG-labeled riboprobes. I alternate between huge background and absolutely nothing. I've basically been following the Jackson protocol. Does anyone have ANY suggestions for troubleshooting??? I'm at my wits end...

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dont know if this will help, but without knowing the specifics of your problems it's hard to comment. I'm posting a cut and paste from my PhD thesis methods on how I did my in-situs for your comparison. ?l = microlitres and ?g = micrograms

All solutions for in situ hybridisation were made up in DEPC-treated H2O. Different glassware was used for pre- and post-hybridisation steps. Proteinase K concentrations needed to be optimised for each probe or tissue type, as well as the hybridisation temperatures for each probe set. Testes were isolated and fixed in 4% paraformaldehyde in PBS at 4°C overnight, dehydrated, embedded in paraffin, and 5 m sections were processed for in situ hybridisation.
2.4.13.1 DNA Template Preparation
When plasmids were used as labelling templates, they were digested with the appropriate enzymes to make a sense and an anti-sense probe, phenol chloroform extracted and ethanol precipitated. Plasmids were then resuspended in 13 l of DEPC H2O and labelled as described below. Alternatively, plasmids were subjected to PCR with T7 and Sp6 in situ hybridisation primers designed to anneal 20 bases on either side of the T7 and Sp6 polymerase promoter sites of p-GEM-T Easy. PCR products were then assessed through DNA electrophoresis, and gel purified using a Wizard SV Gel and PCR Clean-Up System (Promega) according to manufacturer’s instrunctions. Fragments were then quantified spectrophotometrically prior to labelling (described below).
2.4.13.2 Synthesis of DIG-labelled RNA Probes
1-3 g of linearised plasmid or 300 ng of PCR product was labelled using T7 or Sp6 polymerases from Promega. 10 mM DTT, 1X transcription buffer, 40U RNAsin, 2 l DIG RNA labelling mix (Roche Molecular Biochemicals, Mannheim, Germany), 20 U T7 or Sp6 polymerase, and made up to a final volume of 50 l. Reactions were vortexed and pulse spun, then incubated at 37°C for 2 h. 3U of RQ1 DNAse (Promega) was added and each reaction was incubated for a further 20 min at 37°C to remove the DNA template. DNAse digestion was then stopped by adding 1 l of RQ1 DNAse Stop Solution (Promega) and incubating at 65°C for 10 min. Labelled probes were purified by ethanol precipitation (by adding 0.1X v/v 3M sodium acetate pH 5.2 and 2.5X v/v 100% EtOH), centrifuged at 16,000 x g to pellet RNA, and washed in 75% EtOH. Pellets were air-dried and resuspended in 25 l of DEPC H2O. Labelling efficiency was determined by dot-blotting with a control pre-labelled RNA probe (Roche; Section 2.4.13.3).
2.4.13.3 Determination of DIG-Labelling Efficiency
Serial dilutions (10-1-10-5) of each probe and a control probe (at 100 ng/l) were prepared in RNA dilution buffer (DEPC-treated water: 20X SSC: Formaldehyde in the ration 5:3:2) and 1 l aliquots were spotted onto a positively charged nylon membrane (Osmonics, Minnetonka, MN, USA) from dilutions 10-2-10-5. Nucleic acids were UV-crosslinked to membranes for 5 min, and membranes were wetted in Buffer 1A (maleate buffer). Membranes were incubated in Buffer 2A blocking solution (Buffer 1A + 1% BSA) for 20 min at 37°C on a shaking platform. 2 l anti-DIG AP conjugate (Roche) per 10-20 ml buffer was added and incubated on a shaking platform for 25 min at 37°C. Membranes were washed twice in Buffer 1A, then incubated for 2 min in Buffer 3A. Colour detection was then carried out using NBT/BCIP (1:50 dilution of NBT stock solution (Roche) with NBT Buffer) on a shaking platform at room temperature for 5-10 min. Membranes were washed in distilled H2O once colour had developed sufficiently, and probe concentrations were estimated by comparison to the control probe (at 100 ng/l).
2.4.13.4 Slide preparation and Pe-hybridisation
2.4.13.4.1 Coverslip Silinisation
All coverslips used in this protocol were coated in 1% surfasil (Perbio) by dipping three times in acetone, then once in an acetone:1% surfasil solution and dried vertically. This was necessary for all coverslips that were sealed with rubber cement during hybridisation.
2.4.13.4.2 De-paraffinisation of Tissue Sections
De-paraffinisation of sections was carried out by washing 3 x 5 minutes at room temperature in xylene, then twice for 5 min in 100% EtOH. Sections were rehydrated by successive 1 min washes in 90%, 80%, 70%, 60% and 50% EtOH, then in DEPC-H2O for 1 min. From this point onward it was important not to let the sections to dry out, so as to reduce background from non-specific signals.
2.4.13.4.3 Section Permeabilisation and Pre-hybridisation
Once sections were rehydrated they were washed twice in PBS for 5 min, then permeabilised for 15 min in PBS/0.3% Triton-X 100. Slides were washed a further two times in PBS, and proteinase K treated for 30 min at 37°C. The concentration of proteinase K treatment varied depending on each probe and section type, but ranged from 5-20 g/ml (5 – 10 g/ml optimal) in TE buffer. Sections were fixed for 10 min in 4% paraformaldehyde in PBS at 4°C, then washed twice in PBS for 5 min. Sections were then de-acetylated by washing 2 x 5 min in 0.1M triethanolamine buffer (TEA) pH8/0.25% acetic anhydride to reduce non-specific signal. Acetic anhydride was added to each wash immediately prior to use, due to its instability in solution. Slides were then rinsed in PBS, and pre-hybridised for 10-30 min in pre-hybridisation buffer at 37°C. Meanwhile probes were prepared for hybridisation. Each section was hybridised with 50 - 100 ng of probe per section, depending on the intensity of the signal and the abundance of the target mRNA in the tissue. Probes were heat denatured at 80°C for 5 min then cooled on ice for 5 min. Pre-hybridisation buffer was tipped off the sections and sections were overlaid with the probes in 20 l volumes. Sections were covered with silanised coverslips and sealed with rubber cement (Micador Australia Pty Ltd, Box Hill, VIC, 3128) and incubated 12-16 h (overnight) at the optimised annealing temperature.
2.4.13.5 Post-Hybridisation Washes and RNAse Treatment
After hybridisation, the rubber cement was removed from the slides with forceps, and coverslips were gently removed and discarded. Slides were washed in 2X SSC and 50% formamide wash buffer twice for 15 min at 40-60°C (depending on the probe or tissue type). Slides were then washed twice for 10 min in 1X SSC at room temperature.
2.4.13.6 Immunological Signal Detection
Sections were blocked for 30 min – 1 h in Buffer 2B at 4°C. Sections were then incubated with a 1:200 dilution of anti-DIG-AP conjugate (Roche) in Buffer 2B at 37°C in a humid chamber. Slides were washed three times in Buffer 3B for 10 min each at room temperature, then twice in Buffer 4B for 10 min at room temperature.
2.4.13.6.1 Non-Fluorescent Colour Detection (NBT/BCIP)
1:50 dilutions of NBT stock solution (Roche) in NBT buffer were made and sections were overlaid with the solution for 15 -30 min for colour detection. Colour changes were monitored with microscopy until the desired colour was reached, and staining was repeated up to 3 times by washing once in Buffer 3B and overlaying with more NBT solution. Sections were either mounted, or counterstained with Gill’s Haematoxylin. Haematoxylin staining involved covering sections with haematoxylin staining solution and leaving at room temperature for 1 min. Sections were then rinsed under running water for 2 min, mounted in Mowiol, and photographed under bright field microscopy using a Zeiss Axioplan 2 compound microscope.
2.4.13.6.2 Fluorescent Colour Detection (HNPP/Fast Red TR mix)
Fluorescent colour detection was performed for the detection of low expressed signals, using the Roche HNPP Fluorescent Detection Set according to the manufacturer’s instructions. Briefly, 10 l HNPP (10 mg/ml in dimethylformamide) and 10 l Fast red TR solution were mixed with 1 ml of Buffer 4B. This solution was then passed through a 0.2 m nylon syringe filter (Millipore) before use. Sections were overlaid with the HNPP/Fast Red mix, covered with a coverslip, and incubated for 10 min at room temperature. For the detection of single or low copy numbers this step was repeated three times, with slides being washed in Buffer 3B at each interval. Once the desired staining was achieved, slides were washed in distilled water for 10 min. Slides were then counter stained with DAPI (1g/ml in 50% glycerol:50% PBS) for 2 min, then washed once in distilled water for 5 min, and mounted in mowiol. Sections were then visualised using a Zeiss Axioplan 2 compound microscope and selective filters for FITC and DAPI fluorescence.

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