TITLE: RNA in situ hybridization
AUTHORS: Nicholas Leigh, Garrett Dunlap, Kimberly Johnson, Rachelle Mariano, Rachel Oshiro, Alan Y. Wong, Donald M. Bryant, Bess Miller, Jessica L. Whited

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol provides details on tissue harvest and fixation, RNA probe generation, and RNA </span><span style = "font-style:italic;">in situ</span><span> hybridization for use in sectioned axolotl tissue. This protocol was modified from Brent et al, A somitic compartment of tendon progenitors, Cell 2003</span></div></div>

[STEPS]
?. [II. Probe Preparation]
Perform PCR using OneTaq and with T7 and Sp6 primers using pGEM-T easy with probe inserted as template. Do 5+ reactions to ensure ample PCR product. Annealing temperature 40.5°C, extension time time appropriate for length of probe (typically around 30 seconds).   AB1ReagentVolume (μL)25x OneTaq Standard Buffer10310mM dNTP1410μm T7 primer (TAATACGACTCACTATAGGG)1510μm Sp6 primer (ATTTAGGTGACACTATAG)16OneTaq0.2572ng/μL pGEM-T Easy
w/ probe inserted
18Ultrapure&nbsp;H2035.759Reaction Volume&nbsp;50
?. [II. Probe Preparation]
Gel extract PCR product and calculate concentration of gel extracted product.
?. [II. Probe Preparation]
Perform in vitro transcription (3 reactions) with either T7 or Sp6 polymerase depending on anti-sense orientation of probe in pGEM-T Easy. Prepare below reaction and incubate at 37°C for 2 hours.  AB1ReagentVolume (μL)210x Transcription Buffer2310x DIG24Protector RNase Inhibitor0.55Sp6 or T7 RNA polymerase16template (500ng)variable7Ultrapure H20to 20μL
[incubation]
[incubation]
Use RNase-free reagents for all steps and thoroughly RNase Zap all surfaces and pipettes for this step and all subsequent steps of probe preparation.
?. [II. Probe Preparation]
Prepare a gel mold and electrophoresis box by spraying thoroughly with RNase Zap followed by washing out with MilliQ water. In addition, RNase Zap and rinse with MilliQ the interior of a bottle and prepare a 2% agarose gel in this RNase-free bottle.
?. [II. Probe Preparation]
After 2 hour incubation, proceed to RNeasy RNA cleanup with on column DNase I digestion.
?. [II. Probe Preparation]
Combine all three reactions and volume up to 100μL with Ultrapure H20.
?. [II. Probe Preparation]
Add 350μL RLT buffer and mix well.
?. [II. Probe Preparation]
Add 250μL100% ethanol and mix well by pipetting. Proceeded immediately to the next step.
?. [II. Probe Preparation]
Transfer 700μL to a RNeasy Mini spin column that is in a 2mL collection tube, close lid and centrifuge for 15s at 10,000 rpm (8,000g). Discard flow through.
?. [II. Probe Preparation]
Add 350μL Buffer RW1, close lid, and centrifuge for 15s at 10,000 rpm.
?. [II. Probe Preparation]
Combine 10μL DNase I stock solution and 70μL Buffer RDD and mix gently by inverting the tube.
?. [II. Probe Preparation]
Add 80μL of DNase I/Buffer RDD solution directly to the RNeasy column membrane, close lid, and let it sit on the bench for 15min at room temperature.
[incubation]
?. [II. Probe Preparation]
Add 350μL Buffer RW1 to column, close lid, centrifuge for 15s at 10,000 rpm. Discard flow-through.
?. [II. Probe Preparation]
Add 500μL Buffer RPE to column, close lid, and centrigue for 15s at 10,000 rpm. Discard flow-through.
?. [II. Probe Preparation]
Add 500μL Buffer RPE to column, close lid, and centrifuge for 2 min at 10,000 rpm. Discard flow-through.
[centrifugation]
?. [II. Probe Preparation]
Place RNeasy column into a new 2mL collection tube, close lid, and centrifuge at max speed for 1 min.
?. [II. Probe Preparation]
Place RNeasy column into a 1.5mL collection tube (supplied with kit) and add 50μL of Ultrapure H20 directly to the column membrane. Close the lid and centrifuge for 1 min at 10,000 rpm.
?. [II. Probe Preparation]
Remove eluate from collection and re-apply to the column for a second wash.
?. [II. Probe Preparation]
Remove 2ul of eluate and put into a new 1.5mL tube. Store the remainder of the eluate at -80°C until use.
?. [II. Probe Preparation]
Add 8μL of Ultrapure H20 and incubate at 60°C for 10 minutes.
[incubation]
[incubation]
?. [II. Probe Preparation]
Add a clear loading dye (e.g. 40% sucrose) and run on the 2% RNase-free agarose gel to confirm size and quality of product.
?. [III. Hybridization]
Prepare an oven that can accommodate your slide boxes at 65°C.
[oven]
?. [III. Hybridization]
Thaw slides at room temperature (“air-dry”) for 15-20 min.
?. [III. Hybridization]
Rinse slides in 1X DEPC PBS to re-hydrate tissue. This typically requires ~3mL of 1X DEPC PBS
?. [I. Tissue Preparation and Sectioning]
Narcotize animals in 0.1% Tricaine prepared in 40% Holtfreter's. Remove tissue of interest. Deposit directly into 4% PFA prepared in 1X DEPC PBS.
All solutions and tools/bottles should be RNase-free, being prepared with DEPC-treated water or being cleaned with RNase Zap, respectively. 
 
After survival surgeries always keep animals overnight in 40% Holtfreter's supplemented with 0.5% sulfamerazine.
?. [I. Tissue Preparation and Sectioning]
Fix tissues in 4% PFA prepared in 1X DEPC PBS for 1-2 hr at room temperature with gentle rocking or overnight at 4°C with gentle rocking.
?. [I. Tissue Preparation and Sectioning]
Remove 4% PFA/1X DEPC PBS and wash in 1X DEPC PBS by gently rocking tissue in 1X DEPC PBS for 30 min.
?. [I. Tissue Preparation and Sectioning]
Transfer tissue through a sucrose series, starting with 5% sucrose in 1X DEPC PBS, then 10%, 20%, and eventually 30%. Change to higher percent sucrose when tissue sinks.
Once tissue sinks in 30% sucrose in 1X DEPC PBS proceed to the next step. Alternatively, tissue can be rocked overnight in 30% at 4°C.
?. [I. Tissue Preparation and Sectioning]
Add approximately equal volume of OCT compound to the 30% sucrose/1X DEPC PBS. Use a gentle inversion rocker to let it equilibrate for at least 30 minutes.
?. [I. Tissue Preparation and Sectioning]
Prepare a dry-ice/ethanol “bath” and RNase Zap forceps.
?. [I. Tissue Preparation and Sectioning]
Remove tissue from 30% sucrose/OCT solution and move into tissue mold. Fill mold with OCT and orient tissue as desired.
?. [I. Tissue Preparation and Sectioning]
After freezing, move tissue into plastic bags and then store at -80°C until sectioning
?. [I. Tissue Preparation and Sectioning]
Place mold with tissue into dry ice/ethanol bath to freeze OCT.
?. [I. Tissue Preparation and Sectioning]
Section tissue on a cryostat. Sections can be 10-20 microns (usually 16 microns). Collect sections on Superfrost Plus slides.
?. [I. Tissue Preparation and Sectioning]
Air-dry the sections 30 min-2 hrs. Store at -80oC or continue on to hybridization.
?. [III. Hybridization]
Fix slides in 4% PFA DEPC 1X PBS for 10 min.
?. [III. Hybridization]
Rinse in DEPC PBT
?. [III. Hybridization]
Wash twice in DEPC PBT, 5 min. each.
?. [III. Hybridization]
Treat with Proteinase K in DEPC PBS for 10 min. Do this in a bucket with 200 mL DEPC 1X PBS with 1 μg/mL Proteinase K.
?. [III. Hybridization]
Rinse once in DEPC PBT.
?. [III. Hybridization]
Post-fix with 4% PFA 1X DEPC PBS for 5 min.
?. [III. Hybridization]
Three 5 min washes in DEPC PBT.
?. [III. Hybridization]
Acetylation, 10 min, room temperature. Add 625 µl acetic anhydride to 250 mL 0.1M TEA (triethanolamine, prepped with DEPC water) and shake well—use immediately.
?. [III. Hybridization]
Rinse in DEPC PBT.
?. [III. Hybridization]
Wash twice in DEPC PBT, 5 min each.
?. [III. Hybridization]
Rinse in DEPC H2O.
?. [III. Hybridization]
Prepare a humidified slide box. To do this, stand a small slide box on its end and roll a cylinder of paper towels that can fit into the bottom of the box (this will take up about a fifth of the slots). Soak the paper towels in ~25mL of 5X SSC/50% formamide. Set this on your bench with the remaining slots ready to accept the slides.
?. [III. Hybridization]
Take each slide individually and gently let the water wick off onto a Kimwipe. Wipe the edges of the slide with the Kimwipe so that a border of dry slide surrounds the tissue. Continue on to the next step before the tissue sections dry. This step should only take ~1 min/ slide.
?. [III. Hybridization]
Apply the probe. Use 150μl of probe/hybridization buffer for each slide. Gently apply a coverslip-sized piece of plastic bag material (polyethylene) on each slide. Use 2 µl of probe for every 100 µl of hybridization buffer, so here use 3 µl, which should make about 150μl probe. Insert slide into humidified slide box.
Steps 49 and 50 should be repeated for each slide individually. Placing the slides into the slide holder after applying the plastic coverslip.
?. [III. Hybridization]
Seal the box with plastic tape along all the edges, being careful not to let the slides tip forward at all.
?. [III. Hybridization]
Gently transfer the slide box to the prepared 65°C oven. Stand it upright inside. Incubate overnight.
?. [IV. Post-hybridization washes]
The next day, prewarm all the solutions to the appropriate temperature. 65°C: 5X SSC, 2X SSC, 0.2X SSC, 1X SSC/50% formamide. 
  37°C: TNE
?. [IV. Post-hybridization washes]
Once all buffers have come to appropriate temperature remove the coverslips in a bath of 5X SSC (warmed to 65°C) in a glass staining dish that holds slides horizontally. Use a needle to gently break the seal along the edge of the coverslip/slide, which will allow the slips to float to the surface. Discard plastic coverslips. Do this step quickly.
?. [IV. Post-hybridization washes]
Transfer slides to 1X SSC/50% formamide for 30 min at 65°C (in the oven). All steps at 65°C and 37°C should be done in plastic staining dishes.
?. [IV. Post-hybridization washes]
TNE 10 min at 37°C.
[incubation]
[incubation]
?. [IV. Post-hybridization washes]
TNE + RNase A (20μg/mL) for 30 min at 37°C. This is 400 μL of 10 μg/μL stock in 200 mL TNE.
[incubation]
[incubation]
?. [IV. Post-hybridization washes]
TNE 10 min at 37°C (re-use TNE from the first incubation).
[incubation]
[incubation]
?. [IV. Post-hybridization washes]
2X SSC 20 min 65°C
[incubation]
[incubation]
?. [IV. Post-hybridization washes]
0.2X SSC 20 min 65°C.
[incubation]
[incubation]
?. [IV. Post-hybridization washes]
0.2X SSC 20 min 65°C.
[incubation]
[incubation]
?. [V. Antibody Incubation and Detection]
Wash twice in MABT for 5 min. each at room temperature.
?. [V. Antibody Incubation and Detection]
Move slides to a staining tray and add 1mL block/ slide. Block must be left on for a minimum of 1 hour.
?. [IV. Post-hybridization washes]
Prepare block: 20% sheep serum, volumed with MABT, with 2% (w/v) blocking reagent dissolved. Start dissolving block in MABT first, then add the sheep serum. Incubate in a water bath at 55°C, shake intermittently to get it into solution. To determine how much block to make multiply your total number of slides by 2 to determine mL of block. Block solution will be used for the block step as well as the incubation.
?. [V. Antibody Incubation and Detection]
Incubate in a-DIG-AP (1:2000) in 20% HISS/2% blocking reagent(w/v) in MABT (i.e. blocking solution made in step 53), 1mL/slide, overnight, 4°C. Do this in a humidified chamber.
?. [V. Antibody Incubation and Detection]
The next day, rinse once in MABT, then wash three times for 5 min. each in MABT (all room temperature).
?. [V. Antibody Incubation and Detection]
Wash in NTM pH 9.5 for 10 min., room temperature.
?. [V. Antibody Incubation and Detection]
Develop in NTM pH 9.5 + NBT + BCIP. To each mL NTM, add 4.5 μL NBT and 7 μL BCIP. Prepare 1mL/ slide. Once developing keep slides out of direct light.
?. [V. Antibody Incubation and Detection]
Develop until you see the signal without background coming up (different times for different probes). If necessary to develop overnight, keep covered from light in a humidified chamber in the at 4°C. Developing in the cold can help reduce background even when the probe is strong enough to show good signal after a few hours. Prior to moving to 4°C and slides should be washed with NTM. New developing solution should be prepared when incubating at 4°C. This same wash and re-applying new developing solution should be performed if developing continues at room temperature the following day. This can go out for a 2-3 days as long as background doesn't appear.
?. [VI. Post-detection]
Wash in NTM pH 9.5 for 5 min.
[wash]
?. [VI. Post-detection]
Wash in PBS for 5 min.
[wash]
?. [VI. Post-detection]
Fix in 4%PFA/ PBS for 30 min.
[fix]
?. [VI. Post-detection]
Wash: 1X PBS, 1X PBS, water (5 min. each).
?. [VI. Post-detection]
Dry each slide slightly as described earlier in the protocol (step 48). While sections are still slightly wet, apply mounting media (warmed to 65°C) and coverslip. Avoid bubbles.
?. [VI. Post-detection]
Cure the slides in a dry place overnight.
?. [III. Hybridization]
Prepare required buffers. See note for recipes.
SOLUTIONS
 
DEPC water
         In a 4L Erlenmeyer flask, put 3L deionized water in, add 3 mL DEPC, stir overnight (in the hood, with a little foil cap loosely on top). Autoclave the next day. Two 3L flasks should be enough for an in situ.
 
4% PFA in DEPC PBS
         Make a 5X stock = 20% PFA in 5X DEPC PBS. Freeze in aliquots. When defrosting, some PFA/salts might come out of solution. Defrost in a warm bath and shake once in a while. Dilute 1:5 in a Falcon tube to give the correct concentration. Can be repeatedly freeze-thawed.
 
10X PBS (1 L)
         80g NaCl
         2g KCl
         14.4 g Na2PO4
         2.4 g KH2PO4
        
         adjust to pH 7.4 with HCl and bring volume to 1 L with water
 
PBT
         1X PBS +0.1% Tween-20
 
Hybridization Solution
         10 mM Tris pH 7.5
         600 mM NaCl
         1 mM EDTA
         0.25% SDS
         10% Dextran Sulfate 
         1x Denhardt’s (recipe follows)
         200 mg/mL yeast tRNA 
         50% formamide
 **Dissolve Tris and EDTA into DEPC-treated water after DEPC has been autoclaved. Adding Tris and EDTA prior to autoclaving will interfere with the ability of DEPC to eliminate RNases. 
         store at -20oC (in aliquots)
 
100X Denhardt’s solution (100 mL)
         2g BSA
         2g Ficoll (MW 400,000)
         2g Polyvinylpyrolidone (MW 40,000)
         15mL 20X SSC (3x final conc.)
 
         volume to 100 mL
 
20X SSC (1L)
         175.4g NaCl
         88.2g Sodium citrate dihydrate
 
         pH to 7.0, volume, autoclave
 
10X TEA (triethanolamine HCL)
         1M stock pH 8.0
 
TNE
         10mM Tris pH 7.5
         500mM NaCl
         1mM EDTA
 
5X MABT (1L)
         58g Maleic acid
         43.8 NaCl
         volume up to 1L
 
         pH to 7.5 with NaOH 
         filter sterilize
         when ready to use, dilute 1:5 to make 1X MAB, then add Tween to equal 0.1% Tween
 
NTM pH 9.5
         100mM NaCl
         100mM Tris pH 9.5
         50mM MgCl2
 
NBT stock
         50mg/mL in 70% dimethylformamide (store at -20°C, dark)
 
BCIP stock
         25mg/mL in water (store at -20°C, dark)
 
HISS
         Sheep serum, heat inactivated at 56°C for 30 min, store at -20°