TITLE: Vezina Lab RNA in situ hybridization on vibratome sections
AUTHORS: Chad Vezina

[STEPS]
?. [Prior to starting]
Prep stock solutions, make baskets, make probes, section samples.
?. [Day 1]
Prep baskets: a. close cap of tube, affix sticker to cap and label with probe nameb. to facilitate air movement, heat large gauge needle in flame & push through plastic cap to puncture two holes in each basketc. partially submerge baskets in PBSTw in 24-well culture dish
?. [Day 1]
Prep samples:a.sort through tissue section pictures if applicable, choose which sections to use & mark with name of probe to be appliedb.remove samples from 4°C, use spring scissors to remove majority of agarose from those sections that will be used in experimentc.carefully remove any visible debris that may be stuck to sectiond.transfer each section into basket that corresponds with probe to be used
?. [Day 1]
Prepare supplies and preheat solutionsa.remove needed volume of prehybridization solution from –20°C stock & transfer into conical tube, place in hybridization ovenb.prep humidified chamber by adding enough tap water to small plastic storage container to cover bottom, cover container & place in hybridization ovenc.turn hybridization oven on at 60.5°C so everything at desired temperature when reach prehybridization step
?. [Day 1]
Add 2 mL of 6% H2O2 per well of 24-well culture dish, place baskets in wells, incubate 30 min at room temperature with rocking.
?. [Day 1]
Wash 4 x 5 min with PBSTw at RT with rocking.
?. [Day 1]
Add 2 mL proteinase K soln to wells, incubate 12 min at RT with rocking.
?. [Day 1]
Rinse 1 x 5 min with PBSTw at RT with rocking.
?. [Day 1]
Add 2 mL post-fix to wells, incubate 20 min at RT with rocking.
?. [Day 1]
Wash 2 x 5 min with PBSTw at RT with rocking.
?. [Day 1]
Prehybridization step:a. add 1 mL prewarmed prehybridization to wellsb. place culture dish in humidified chamber & coverc. incubate at least 1 hr in 60.5°C hybridization oven with rocking
?. [Day 1]
Hybridization stepa.for each probe, calculate vol needed to yield 0.325 mgb.add appropriate vol of each probe to prehybridization soln in corresponding wellc.replace culture dish in humidified chamberd.incubate overnight in 60.5°C hybridization oven with rocking
?. [Day 1]
Prepare and preheat solution for Day 2a. remove needed vol of Solution 1 from –20°C stockb. add appropriate vol of 10% SDS (may want to wait until Solution 1 warms to RT) c. heat in hybridization oven overnight so ready for Day 2 wash steps
?. [Day 2]
Wash 3 X 30 min with Soln 1 in 60.5°C hybridiation oven with rocking
?. [Day 2]
Prepare and preheat solutions:a.once samples in first Soln 1 wash, remove enough Soln 2 from RT stock to make Soln 1 / Soln 2 mix, warm in 60.5°C hyb ovenb.remove enough Soln 2 from RT stock for one wash, add RNase, warm at 37°Cc.remove needed vol of Soln 3 from –20°C stock, set aside enough for one wash at RT, start warming rest of Soln 3 in 60.5°C hyb oven
?. [Day 2]
Wash 1 X 10 min with 50% Soln 1 / 50% Soln 2 in 60.5°C hyb oven with rocking
?. [Day 2]
Wash 4 X 10 min with Soln 2 at RT with rocking
?. [Day 2]
Add 2 mL RNase soln to wells, incubate 15 min at 37°C
?. [Day 2]
Wash 1 X 10 min with Soln 2 at RT with rocking
?. [Day 2]
Wash 1 X 10 min with Soln 3 at RT with rocking
?. [Day 2]
Wash 2 X 1 hr with Soln 3 in 60.5°C hyb oven with rocking
?. [Day 2]
Begin thawing aliquots of Tissue Blocking (TB) & Antibody Dilution (AD) buffers** you do not need to thaw AD buffer for every run because antibody soln can be reused several times—check fridge for antibody soln before thawing AD buffer**
?. [Day 2]
Wash 3 X 10 min with TBSTw at RT with rocking
?. [Day 2]
Begin thawing aliquot of Antibody Absorption (AA) buffer**you do not need to thaw AA buffer for every run because antibody soln can be reused several times—check fridge for antibody soln before thawing AA buffer**
?. [Day 2]
Blocking step:a.add 2 mL TB buffer to wellsb.incubate at least 2 hr at RT with rockingc.if fresh antibody soln is needed, add 3.3 mL anti-DIG antibody per 600 mL aliquot of AA buffer & incubate at least 2 hr at 4°C with rocking
?. [Day 2]
Antibody step using fresh antibody soln [to reuse antibody soln, see step 27]a.spin AA buffer + antibody at 10,000 rpm for 1 min to collect embryo powderb.carefully draw off supernatant, add entire volume to AD buffer & mixc.add 2 mL AD buffer + antibody to wellsd.place culture dish in humidified chambere.incubate overnight at 4°C with rocking
?. [Day 2]
Antibody step reusing antibody solna.remove antibody soln from 4°C, add 1 mL soln to wellsb.place culture dish in humidified chamberc.incubate overnight at 4°C with rocking
?. [Day 3]
Remove antibody soln a.  if soln is to be reused, collect total vol in conical tube & store at 4°C b.  following initial use only, add 1 µL of 0.2 M sodium azide soln per 1 mL antibody soln to prevent contamination c.  clearly mark tube with number of times soln used, antibody soln should be discarded after three applications
?. [Day 3]
Wash 8 X 10 min with TBSTw + levamisole at RT
?. [Day 3]
Remove sections from baskets, use forceps to separate sections & remove any visible debris, transfer into clean microcentrifuge tubes according to probe
?. [Day 3]
Wash 1 X 10 min with NTMT + levamisole at RT with rocking
?. [Day 3]
Detection step a.  prep 40% BM Purple substrate in NTMT + levamisole, add 1 mL per tube b.  wrap tubes in foil, incubate at RT with rocking while color develops (color development time ranges from several hours to several days) c.  change BM Purple + levamisole soln as needed (substrate will precipitate over time). d.  Switch over to 100% BM purple (Containing a final concentration of 2mM levamisol and 0.1% Tween-20) if staining is not apparent after about 20 hours. e.  Once color fully developed, wash 2 X with NTMT + levamisole at RT
?. [Day 3]
Bleaching step a.  post-fix in 1 mL 4% PFA at least overnight, rinse with 1 mL PBSTw b.  make 3% H2O2 soln, add 1 mL per tube, rock at RT for at least 30 min c.  wash 1 X 10 min with 1 mL PBSTw at RT with rocking d.  post-fix in 4% PFA, image samples & store at 4°C
?. [Solution Recipes]
PBSTw:1X PBS + 0.1% Tween 20, add 1 mL of 0.2 M sodium azide per 1 mL PBSTwto prevent contaminating growth, sterile filter to remove insolubles/contaminants
?. [Solution Recipes]
0.2 M sodium azide: Dissolve 1.3 g sodium azide in 100 mL double-distilled H20, pH to 7.6 (note: quite sensitive to pH change so need very little NaOH to adjust)
?. [Solution Recipes]
6% H2O2: 1 mL 30% H2O2per 4 mL PBSTw
?. [Solution Recipes]
Proteinase K: 0.25 mL 20 mg/mL prot K per 1 mL PBSTw
?. [Solution Recipes]
Post-fix: 8 mL 25% glutaraldehyde per 1 mL 4% PFA
?. [Solution Recipes]
ABC1stock solutionfinal conc.for 1000 mL2100% formamide  50%  500  mL320X SSC  5X  250  mL4Blocking reagent  1%  100 mL of a 10% solution in Maleic acid buffer, pH 7.5510 mg/mL yeast tRNA10 mg/mL1 mL610 mg/mL heparin10 mg/mL1 mL7dH2O to vol.  --  to 1000 mL Aliquot 50 mL volumes into conical tubes & store at –20°C
ABC1stock solutionfinal conc.for 1000 mL2100% formamide  50%  500  mL320X SSC  5X  250  mL4Blocking reagent  1%  100 mL of a 10% solution in Maleic acid buffer, pH 7.5510 mg/mL yeast tRNA10 mg/mL1 mL610 mg/mL heparin10 mg/mL1 mL7dH2O to vol.  --  to 1000 mL
?. [Solution Recipes]
Solution 1:  ABC1stock solutionfinal concfor 500 mL2100% formamide  50%  250 mL320X SSC  5X  125 mL4  dH2O    --  75 mL510% SDS  1%  50 mLMix formamide, SSC & dH2O according to above & store at –20°CDO NOT add SDS to freezer stock soln because SDS will precipitate in the coldPrior to using Soln 1 for ISH, add 1 mL of 10% SDS per 9 mL Soln 1 stock
ABC1stock solutionfinal concfor 500 mL2100% formamide  50%  250 mL320X SSC  5X  125 mL4  dH2O    --  75 mL510% SDS  1%  50 mL
?. [Solution Recipes]
Solution 2  ABC1stock  solnfinal  concfor  500 mL21M Tris-HCl, pH 7.510mM5 mL35M NaCl0.5M50  mL4100% Tween 20  0.1%  0.5  mL50.2  M sodium azide0.2mM0.5 mL6  dH2O    --  444 mLSterile filter to remove insolubles/contaminants, store at RT
ABC1stock  solnfinal  concfor  500 mL21M Tris-HCl, pH 7.510mM5 mL35M NaCl0.5M50  mL4100% Tween 20  0.1%  0.5  mL50.2  M sodium azide0.2mM0.5 mL6  dH2O    --  444 mL
?. [Solution Recipes]
RNase:5 mL RNase (50 mg/mL) per 1 mL Soln 2
?. [Solution Recipes]
Solution 3  ABC1stock solnfinal  concfor  500 mL220X  SSC  2X  50  mL3100%  formamide  50%  250  mL4  dH2O    --  200  mLStore at –20°C
ABC1stock solnfinal  concfor  500 mL220X  SSC  2X  50  mL3100%  formamide  50%  250  mL4  dH2O    --  200  mL
?. [Solution Recipes]
Tissue blocking (TB) buffer  ABC1stock  solnfinal  concfor  500 mL210X  TBS  1X  50  mL3100%  sheep serum  10%  50  mL410%  blocking reagent  1%  50  mL5  BSA    1%  0.5 g6dH2O  to vol.  --  to  500 mL7100%  Tween 20  0.1%  0.5  mLMix TBS, serum, blocking reagent, BSA & dH2O according to aboveFilter through #2 Whatman filtersAdd Tween 20, aliquot 6 mL volumes into conical tubes & store at –20°C
ABC1stock  solnfinal  concfor  500 mL210X  TBS  1X  50  mL3100%  sheep serum  10%  50  mL410%  blocking reagent  1%  50  mL5  BSA    1%  0.5 g6dH2O  to vol.  --  to  500 mL7100%  Tween 20  0.1%  0.5  mL
?. [Solution Recipes]
Antibody dilution (AD) buffer  ABC1stock  solutionfinal  concfor  500 mL210X  TBS  1X  50  mL3100%  sheep serum  5%  25  mL410%  blocking reagent  1%  50  mL5  BSA    1%    0.5
  g  6dH2O  to vol.  --  to  500 mL7100% Tween 20  0.1%  0.5  mLMix TBS, serum, blocking reagent, BSA & dH2O according to aboveFilter through #2 Whatman filtersAdd Tween 20, aliquot 6 mL volumes into conical tubes & store at –20°C
ABC1stock  solutionfinal  concfor  500 mL210X  TBS  1X  50  mL3100%  sheep serum  5%  25  mL410%  blocking reagent  1%  50  mL5  BSA    1%    0.5
  g  6dH2O  to vol.  --  to  500 mL7100% Tween 20  0.1%  0.5  mL
?. [Solution Recipes]
TBSTw:1X TBS + 0.1% Tween 20, add 1 mL of 2 M sodium azide per 1 mL TBSTw to prevent contaminating growth, sterile filter to remove insolubles/contaminants
?. [Solution Recipes]
Antibody absorption (AA) buffer  ABC1stock solnfinal concfor 20 mL21X  TBSTw  --  17  mL3100% sheep serum  5%  1  mL410%  blocking reagent  1%  2  mL5  BSA    1%  0.2 g6embryo  powder  --  0.12 gShake at 4°C for 30 min to rehydrate embryo powderAliquot 600 mL volumes & store at –20°CSheep serum (must be heat-inactivated before use)To heat inactivate: thaw new bottle of serumincubate 70°C for 30 minaliquot & store at –20°C
ABC1stock solnfinal concfor 20 mL21X  TBSTw  --  17  mL3100% sheep serum  5%  1  mL410%  blocking reagent  1%  2  mL5  BSA    1%  0.2 g6embryo  powder  --  0.12 g
?. [Solution Recipes]
10% Blocking reagent  ABC1stock solnfinal  concfor  100 mL2maleic  acid100  mM1.2  g35 M NaCl  150
  mM  3 mL4dH2O  to vol.  --  to 100 mL5Blocking  reagent  10%  10  gMix maleic acid, NaCl & dH2O according to above, pH to 7.5 (note: strong buffer so difficult to pH, try using solid NaOH pellets to raise pH initially)Add blocking reagent, microwave briefly to aid solubility (avoid boiling over, soln will be cloudy & viscous so watch carefully to ensure blocking reagent is completely in soln)Aliquot 10 mL volumes into conical tubes & store at –20°C
ABC1stock solnfinal  concfor  100 mL2maleic  acid100  mM1.2  g35 M NaCl  150
  mM  3 mL4dH2O  to vol.  --  to 100 mL5Blocking  reagent  10%  10  g
?. [Solution Recipes]
2 M Levamisole: Dissolve 4.82 g levamisole in 7 mL double-distilled H2O (total vol should equal 10 mL), aliquot 200 mL volumes & store stocks at –20°C
?. [Solution Recipes]
TBSTw + levamisole: 1 mL of 2 M levamisole per 1 mL 1X TBSTw
?. [Solution Recipes]
NTMT + levamisole (inhibits endogenous alkaline phosphatases):  ABC1stock  solnfinal  concfor  500 mL21  M Tris-HCl, pH 9.5100  mM50  mL35  M NaCl100  mM10  mL41  M MgCl250 mM25  mL50.2  M sodium azide  0.2
  mM  0.5  mL6  dH2O    --  414.5  mL7100%  Tween 20  0.1%  0.5  mL82  M Levamisole  2
  mM  0.5 mLMix Tris, NaCl, MgCl2, sodium azide & dH2O according to aboveSterile filter to remove insolubles/contaminants, store at RTDO NOT add Tween or levamisole to stock solnPrior to using NTMT for ISH, add 1 mL of100% Tween 20 and 1 mL of 2 M levamisole per 1 mL NTMT stock
ABC1stock  solnfinal  concfor  500 mL21  M Tris-HCl, pH 9.5100  mM50  mL35  M NaCl100  mM10  mL41  M MgCl250 mM25  mL50.2  M sodium azide  0.2
  mM  0.5  mL6  dH2O    --  414.5  mL7100%  Tween 20  0.1%  0.5  mL82  M Levamisole  2
  mM  0.5 mL
?. [Solution Recipes]
3% H2O2:1 mL 30% H2O2per 9 mL PBSTw
?. [Solution Recipes]
Embryo powder:a. Collect embryo tissue & store at –80°C until ready to make powderb. Place small amounts of frozen tissue into mortar, add liquid nitrogen & use pestle to grind tissue into powder (add more liquid nitrogen as needed)c. Use dounce homogenizer to grind tissue further: combine embryo powder with 4 volumes of acetone in homogenizer, homogenize with several strokes of dounce until ground to fine powderd. Transfer acetone + powder to 15 mL glass screw-top vials, make sure powder is in 4 volumes of acetone & shake overnight at 4°Ce. Wrap vials in 2 folded paper towels, place wrapped vials in tabletop centrifuge adaptors for 50 mL conical tubes & spin at 5000 rpm for 10 min at RTf .Remove & discard supernatantg. Add 4 volumes fresh acetone to powder in vials & shake 2 hr at 4°Ch. Spin according to abovei. Remove & discard supernatantj. Spread pellet onto #2 Whatman filter paper & allow to air dry in hoodk. Once dry, use mortar and pestle to grind pellet into fine powderl. Store powder in tightly sealed glass vial at 4°Cm. The approximate yield is 50 mg powder per 1 g embryo wet weight.