IN SITU HYBRIDIZATION DATA PRODUCTION
Overview
The processes and protocols used by the Marmoset Gene Atlas (the Atlas) weredesigned based upon the methods developed by Dr. Cliff Ragsdale’s laboratory
at the University of Chicago (https://ragsdalelab.uchicago.edu/), (Grove EA. etal., 1998: Agarwala A. et al., 2001: Sanders TA. et al., 2002). The data
production laboratory (the Shimogori Lab) is located at RIKEN Brain Science
Institute in Wako, Saitama, Japan. ISH is performed by some modification to apreviously reported method (Mashiko H. et al., 2012).

Methods
1. Sample collection
Early postnatal marmosets are anesthetized with a lethal dose of pentobarbitone(100 mg/kg), and after three failed attempts to elicit a foot withdrawal reflex theanimals are transcardially perfused with 4% paraformaldehyde in PBS. Brains
are removed and fixed in 30% sucrose/4% PFA in PBS for several days.

2. Sectioning
Brains are sectioned in the coronal plane on a freezing microtome at 28 µm. One
hemisphere of the brain is marked with DiI solution (dissolved in 4M
Sucrose/PBS) and each section is individually mounted on slides and processedfor ISH.

3. Preparation of DNA template for cRNA synthesis
Primers for polymerase chain reaction (PCR) targeting common marmoset
genes are designed with (primer3, ver. 0.4.0) software based on the common
marmoset genome sequence. Total RNA is isolated from frozen embryonic andpostnatal marmoset brains and collected in Trizol reagent (Invitrogen, Carlsbad,CA). cDNA is generated from total RNA using prime script 1st strand cDNA
synthesis kit (Takara, Shiga, Japan) according to the manufacturer’s
recommended procedure.

4. Preparation of linearized templates:
Prepare plasmid DNA using Qiagen miniprep kit or equivalent. Following
plasmid preparation, all steps should be performed using RNase-free solutions.
Digest 5-10 µg of template DNA to completion using restriction enzyme of choice.
Confirm completeness of digestion using gel electrophoresis. Following
digestion, add 5 µL protease K (19 mg/mL stock) and 5 µL of 10% SDS andadjust total volume to 110 µL with ddH2O and incubate at 37ºC for 15 minutes.
Extract with 110 µL phenol/chloroform/isoamyl alcohol (25:24:1) once and 1101µL of chloroform/isoamyl alcohol (24:1) twice. Precipitate DNA by adding 200µLof 100% EtOH and 4µL of 5M NaCl. Store at -20ºC at least 30 minutes.
Centrifuge for 15 minutes at 4ºC at maximum speed to collect pellet, then wash
once with 200 µL 70% EtOH. Air-dry the pellet and resuspend to 0.5 ~ 1.0 µg/µLin TE.

5. Generation of cRNA probe:
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Following purification of template DNA, synthesize probe by mixing thefollowing components in the order indicated. Use RNase-free aerosol tips
and RNase-free solutions for all procedures: 2 µL 10x RNA polymerase
buffer, 2 µL 10x DIG NTP mix, 11 µL ddH2O, 1 µL 0.1M DTT, 1 µL RNaseinhibitor, 1 µL RNA polymerase (T7, T3 or Sp6) to 18 µL final volume.
Generate a master mix using these specifications when screening multipleprobes. Finally, add 1 µg of template in 2 µL TE. This reaction can be
performed in RNAse-free Eppendorf tubes or in 96 well RNAse-free PCR
platesIncubate 3 hours at 37ºC.
Precipitate by adding 180 µL ddH2O, 400 µL 100% EtOH, and 8 µL 5MNaCl. Vortex at maximum speed for 5 seconds. If probe synthesis isperformed in 96 well plate format, transfer the product to RNAse free
Eppendorf tubes prior to precipitation.
Store at -20ºC for at least 1 hour.
Centrifuge for 15 minutes at 4ºC at maximum speed to collect pellet. Wash
pellet once in 200 µL 70% EtOH. Air-dry the pellet for 5-10 minutes anddissolve in 100 µL TE. Run denaturing gel with RNA size marker to checkprobe yield and integrity.

6. Pre-hybridization treatment and hybridization

Fix in fresh 4% paraformaldehyde in PBS (aka PFA) for 15 minutes at roomtemperature.
Wash 3x 5 minutes in DEPC-PBS at room temperature. In this and all
subsequent washes, place mailers on rotating platform to ensure goodmixing.
Incubate in Proteinase K solution (1µg/mL) at 37°C for 30 minutes. [Add 3µl of 17 mg/mL Proteinase K stock per 50 ml 100 mM Tris-HCl pH 8, 50
mM EDTA pH 8.0.] Use a fresh aliquot of Proteinase K every 2 weeks.
Fix in PFA for 15 min.
Wash 3x 5 minutes in DEPC-PBS at room temperature.
Hybridization: add 90~100 µL of DIG and/or FL-labeled probe to 14 mL
hybridization solution per mailer. Mix well. Place slides into mailer andfill empty slots with RNase-free dummy slides. Incubate at 72°C overnightin dry oven or water bath.

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7. Post-hybridization and color development
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Transfer slides from hybridization mailer to mailers containing Solution X.
Wash 3x 45 minutes in Solution X at 72°C. Use of rotating platform isunnecessary for this step.
Wash for 3x 15 min in TBST at room temperature.
Block for 1 hour in 10% lamb serum in TBST at room temperature (orovernight at 4°C).
Incubate in pre-absorbed alkaline phosphatase-conjugatedanti-digoxygenin antibody [1:5000 in 1% lamb serum in TBST] for 2 hours atroom temperature or overnight at 4°C.
Wash 3x 15 min in TBST at room temperature to remove excess antibody.
Wash for 15 min in freshly prepared NTMT at room temperature.
Begin color reaction by adding 35µL NBT (100mg/mL in 100% DMF) and35µL BCIP (50mg/mL in 100% DMF) per 10 mL NTMT. Incubate at roomtemperature in the dark. Continue color reaction overnight at room
temperature or 4°C as appropriate.
When color has developed, rinse quickly in NTMT buffer, then wash 3x 15
min in TBST at room temperature to remove background. Stop reaction byadding TE Stop Buffer.

8. Cover slip and image acquisition
After color reaction is completely stopped in TE Stop Buffer, slides aredehydrated with ethanol, tissue clearing is performed using Histo-Clear (NationalDiagnostic. GA) and cover slips are placed using Eukitt (Electron MicroscopyScience. PA). Images of whole slides are collected by scanning with the LeicaSCN400 Slide Scanner.

9. Solutions

4% paraformaldehyde (PFA): 45 mL ddH2O, 4g paraformaldehyde
To prepare, heat to 60-70ºC, add 1 drop 10N NaOH, stir to dissolve. Once
PFA has fully dissolved, add 5mL DEPC-treated 10xPBS, pH 7.5. Sterile
filter and store on ice.
20x SSC, pH 4.5 (1L): Add 175.9g NaCl and 88.2g
Na3(C3H5O(COO)).2H20) to 800 mL of ddH2O. Adjust pH to 4.5 withconcentrated HCl. Adjust volume to 1000 mL final.
0.2M Phosphate buffer, pH 7.5: Dissolve 3.84g of KH2PO4 and 45.1 g
Na2HPO4 into 800 mL ddH2O. Adjust pH to 7.5 with HCl. Adjust volumeto 1000 mL final.
Hybridization Solution: 50% formamide, 5 x SSC (pH 4.5), 1% SDS, 500µg/mL tRNA, 200 µg/mL acetylated BSA, 50 µg/mL heparin. For 500mL ofhybridization solution: 250 mL ultrapure formamide, 125 mL 20xSSC (pH34.5), 50 mL 10% SDS, 2.0 mL tRNA (125 mg/mL in DEPC ddH2O), 4.0 mLacetylated BSA (25 mg/mL in DEPC ddH2O), 2.5 mL heparin (10 mg/mL in
DEPC ddH2O), and 68.5 mL DEPC ddH2O.
Solution X: 2 x SSC (pH 4.5), 50% formamide, 1% SDS For 500 mL ofSolution X: 50 mL 20xSSC (pH 4.5), 250 mL ultrapure formamide, and 50
mL 10% SDS, and 150 mL DEPC ddH2O.
NTMT: 100 mM NaCl, 100 mM Tris-HCl (pH 9.5), 50 mM MgCl2, 1% Tween
20. For 250 mL of NTMT: Combine 12.5 mL 2M NaCl, 25 mL 1M Tris-HCl(pH9.5),12.5 mL 1M MgCl2, 2.5 mL Tween-20, 197.5 mL ddH2O.
TE Stop Buffer. 10 mM Tris-HCl (pH 7.5). 10 mM EDTA (pH 8.0)

10. References
Grove EA, Tole S, Limon J, Yip L, Ragsdale CW. (1998) The hem of theembryonic cerebral cortex is defined by the expression of multiple Wnt genes
and is compromised in Gli3-deficient mice. Development 125:2315-25.
Agarwala S, Sanders TA, Ragsdale CW. (2001) Sonic hedgehog control of sizeand shape in midbrain pattern formation. Science. 291:2147-50.
Sanders TA, Lumsden A, Ragsdale CW. (2002) Arcuate plan of chick midbrain
development. J Neurosci. 22:10742-50.
Mashiko H, Yoshida AC, Kikuchi SS, Niimi K, Takahashi E, Aruga J, Okano H,
Shimogori T. (2012) Comparative anatomy of marmoset and mouse cortex fromgenomic expression. J Neurosci. 32:5039-53.
Shimogori T and Blackshaw S. In Situ Hybridization Methods. PartIII, 10: 207220. Springer Protocols. Human Press.

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