Free-floating section In Situ Hybridization (ver.1.3)

Transcription

1 Free-floating section In Situ Hybridization (ver.1.3) Written by Akiya Watakabe March 3, 2004 Revised Aug, 30, 2007 Ref: 1) Liang, F., Hatanaka, Y., Saito, H., Yamamori, T. & Hashikawa, T. (2000) J Comp Neurol 416, ) Tochitani, S., Liang, F., Watakabe, A., Hashikawa, T. & Yamamori, T. (2001) Eur J Neurosci 13, ) Komatsu, Y., Watakabe, A., Hashikawa, T., Tochitani, S. & Yamamori, T. (2005) Cereb Cortex 15, ) Watakabe, A., Ohsawa, S., Hashikawa, T., & Yamamori, T. (2006) J Comp Neurol 499, ) Watakabe, A., Ichinohe, N., Ohsawa, S., Hashikawa, T., Komatsu, Y., Rockland, K., & Yamamori, T. (2007) Cerebral Cortex17, Adapated by Yusuke Komatsu and Akiya Watakabe Dr. Tetsuo Yamamori s lab National Institute for Basic Biology, Okazaki, Japan. watakabe@nibb.ac.jp

2 0. Reagents: <NOTES> In this protocol, letter u is used to represent micro, to minimize confusion due to font change. Thus, ug and ul means microgram and microlitter, respectively. >4% PFA (paraformaldehyde)/0.1m PB >0.1M PB (phosphate buffer, ph7.0) >0.75% Glycine/0.1M PB >0.3% Triton X100/0.1M PB >PK Buffer (0.1M Tris.HCl (ph8.0), 50 mm EDTA) >Acetylation Buffer Mix 167 ml WATER, 2.26 ml triethanolamine and 0.3 ml HCl Add 10 ul acetic anhydride into 4 ml of acetylation buffer just before use >Maleic acid buffer (0.1 M Maleic acid, 0.15M NaCl, ph7.5) >10 % Blocking reagent (10 % Blocking reagents (Roche# ) in Maleic acid buffer) Autoclave to completely dissove the reagents. Better be kept frozen aliquoted for long-term storage >2% NLS(N-lauroylsarcosine) warm to dissolve well (It remains cloudy even after warming) >Hybridization sol.(for ~50 ml) 20xSSC (3M NaCl, 0.3M Na-citrate) 12.5 ml 10 % Blocking reagent 10 ml Formamide (molecular biology grade) 25 ml 2% NLS 2.5 ml 10 % SDS 0.5 ml >2xSSC/50% formamide/0.1% NLS >RNase Buffer (10 mm Tris-HCl, ph 8.0, 1 mm EDTA, 0.5 M NaCl) >2xSSC /0.1% NLS >0.2xSSC /0.1% NLS

3 >TS7.5 (0.1 M Tris-HCl, ph7.5, 0.15M NaCl) >1% Blocking Sol (1:10 Dilute 10 % Blocking reagent with TS7.5) >TNT (TS7.5, 0.05 % Tween 20) >TS9.5 (0.1 M Tris-HCl, ph9.5, 0.1M NaCl, 50 mm MgCl2) >Proteinase K (Roche: PCR grade # ) >RNase (RNaseA: sigma #R4642) Anti-Digoxigenin-AP, Fab fragment (Roche) NBT/BCIP solution (Roche) PBS-EDTA: PBS with 10 mm EDTA Mounting media:mix 0.1 % gelatin, 40 % ethanol with PBS at 1:1 ratio >cryoprotectant solution (for storing sectioned tissues) 30 % glycerol, 30 % ethylene glycol, 40 % 0.1M PBS (mix 300 ml glycerol, 300 ml ethylene glycol and 400 ml 0.1M PB containing 3.4g NaCl) <Coating the slideglass with gelatin> Coating solution> Dissolve 1.25 g of gelatin, g of Chromium Potassium sulfate in pre-warmed MilliQ water to make 250 ml of solution. Filtrate though wattman filter paper. Place clean slideglasses in a holder. Put them into the coating solution for 10 sec and dry at room temp for a few days. You can store the coated slideglasses for months and years.

4 1. Overview: <DAY1> Cut the section. Postfix in 4% PFA <DAY2> 4C overnight 0.1M PB 10 min x % Glycine/0.1M PB 15min x 2 0.3% Triton X100/0.1M PB 20 min 0.1M PB 5 min PK treatment 37C 30 min Acetylation Buffer 10 min 0.1M PB 10 min x 2 Hybridization sol. 60C 1 hour Hybridization sol.+rna probe 60C overnight <DAY3> 2xSSC/50% formamide/0.1% NLS 60C min x 2 RNase Buffer 5min RNase (20ug/ml) 37C 30min 2xSSC /0.1% NLS 37C min x 2 0.2xSSC/0.1% NLS 37C min x 2 TS7.5 5min 1% Blocking reagent 1hour 1/1000 anti-dig-ap in 1% Blocking reagent 2~5 hours at room temp or overnight at 4C TS7.5, 0.05% Tween20 15 min x 3 TS min 1/50 NBT/BCIP in TS min~30 hours Stop reaction in PBS containing 10 mm EDTA Mount on gelatin coated slideglass

5 2. General guidance <Handling samples> It is most important to ensure the RNase-free environment for dealing with RNA. Ware gloves before hybridization step. RNase is very stable and is not completely denatured even by autoclaving. Use RNase in a restricted area and be careful not to spread the RNase that is often used for molecular biological experiments. We routinely use absolve (NEN) cleaner to wipe benchtops and clean the plates used after RNase-treatment. <glasswares etc> Disposable mutiwell plate (6, 12 and 24-well plates) is used for pretreatment and wash steps. Plates used for pretreatment can be reused after washing and rinsing with DEPC-treated water. For handling tissue sections, heat the tip of a Pasteur pipette to melt and make a hook. <Specificity of hybiridization> Specificity of the hybiridization pattern is always a matter of concern. The must control is the sense probe. But bear in mind that the absence of hybiridization signals with the sense probe does not ensure the specificity of the antisense probe. Making two kinds of antisense probes with non-overlapping sequence would be a good confirmation. If they are identical, you can mix the probes to make the signal stronger. Avoid GC-rich sequences, because they tend to produce non-specific hybridization. In my opinion, the best evidence for the specificity is the pattern itself. If you see a characteristic pattern, it is easier to believe the specificity of ISH. If not, you need to be very careful about the specificity. The non-specific hybridization is not necessarily uniform. Some structures tend to produce higher background signals such as the dendate gyrus of hippocampus.

6 <DIG-probe> Contamination of free DIG-nucleotides can be a cause of high background. Ethanol precipitation alone cannot efficiently remove the free nucleotides. We usually use a spun column (Probe quant G-50: amerciam-pharmacia) before ethanol precipitation. The standard length of RNA probe in my experiments are nt. For a longer probe (>800 nt), hydrolysis may improve the signal intensity. When I used a cdna probe spanning 2.6kb, hydrolysis made a big difference in hybridization intensity. Below is the protocol for hydrolysis. Reagents: 0.25M NaHCO3 0.25M Na2CO3 Hydrolysis buffer Mix 2 ml of NaHCO3, 3 ml of Na2CO3 and 5.75 ml DEPC-treated water. (This is expected to be 0.1M Na-CO3 buffer, ph 10.2) Protocol> >In vitro transcription in 20 ul. >Add 180 ul Hydrolysis buffer and incubate at 60C for appropriate time. Probe length (kb) Time (min) > Immediately place the tube on ice and add 1 ul of glacial acetic acid. > Add 20 ul of 3M NaOAC, 500 ul EtOH and mix well. > Immediately spin at rpm for 15 min. > Discard the sup. Dissolve the pellet to 50 ul STE. > ProbeQuant G-50 spun column. > Ethanol precipitate.

7 3. Detailed protocol: <DAY1> >> Preparing tissue sections. Perfusion fix is desired. Tissue blocks are postfixed in 4% PFA/0.1M PB (ph 7.0), for several hours, then immersed in 30 % sucrose /0.1M PB (ph 7.0), for cryoprotection and then frozen just before use or stored frozen at -80C. Use DEPC-treated water to make sucrose solution. Tissue sections are cut in a sliding microtome and each section is collected by a paintbrush into either 0.1M PB (ph 7.0) or 4% PFA/0.1M PB (ph 7.0). Unless the buffer is contaminated by RNase, tissue sections can be stored 1~2 weeks for ISH. For long-term storage of sections, store them in cryoprotectant solution (see reagents) and keep in 20C freezer. >> Postfix Postfix the tissue sections in 4% PFA/0.1M PB (ph 7.0) at 4C overnight. PFA needs to be fresh. I usually use frozen-stocked PFA melted just before use. PFA kept at 4C for a few days is not good. <DAY2> >> Pre-PK wash 0.1M PB 10 min x % Glycine/0.1M PB 15min x 2 0.3% Triton X100/0.1M PB 20 min 0.1M PB 5 min Wash the tissue sections in multiwell dishes with gentle agitation in appropriate buffers as above. Use a fire-molded Pasteur pipette to move the sections. >> PK treatment 37C for 30 min with gentle agitation Proteinase K treatment is very important. The optimal condition needs to be

8 determined empirically. Generally speaking, more PK, more signals but the sections become more fragile. I usually use 0.5~3.3 ug PK/ml for adult mouse brains and 5-10 ug PK/ml for adult monkey brains. The concentration should be lower for embryonic brains. Stronger PK treatment is possible by adding % SDS to the PK buffer. This may increase the signal intensity but the sections may become very fragile. >>Acetylation 10 min Add acetic anhydride just before use. Quickly swirl and put in sections. Acetylation Buffer 10 min 0.1M PB 10 min x 2 >> Prehybridization Place the sections in a pre-heated hybridization sol. Keep at 60C for 1 hour (45 min + 15 min, see below) with gentle agitation. >> Hybiridization Heat the DIG probes in hybridization buffer at 80C for 5 min and quickly place the tubes in a heat oven and keep at 60C until used. I usually use ug of DIG probes /ml hybridization sol (final conc). (This is a large probe excess. Depending on the strength of the signal, you can reduce the amount of the probe to maybe 0.1 ug/ml or less without so much signal reduction.) Move the sections to a well containing fresh pre-warmed hybridization sol., keep min at 60 C and quickly add the heatdenatured DIG probe in hybridization sol to each well. Swirl and hybridize overnight at 60C with gentle agitation. To avoid drying, place the mutiwell dish in a humid chamber with a wet paper towel. Don t agitate too hard. The PK treated sections are very fragile. The hybiridization temperature needs to be determined empirically. For some probes, hybiridization at as high temperature as 72C is required to reduce non-specific binding. If specific hybiridization can be done at lower temp such as 50C, the morphology is

9 usually better. But in my hand, hybiridization at 60C was somehow generally more sensitive than at 50C. <DAY3> >> Post-hybridization wash 2xSSC/50% formamide/0.1% NLS 60C 20 min x 2 RNase Buffer 5min RNase (20ug/ml) 37C 30min 2xSSC /0.1% NLS 37C 20 min x 2 0.2xSSC/0.1% NLS 37C 20 min x 2 TS7.5 5min Wash the sections in 2xSSC/50% formamide/0.1% NLS pre-warmed to 60C. Use the same temperature as was used for hybridization. Wash briefly in RNase buffer and treat the sections with RNase. After RNase treatment, wash sections twice in 2xSSC /0.1% NLS and twice in 0.2xSSC/0.1% NLS. After this stringent wash, equilibrate the sections in TS7.5. <Note> Beware of RNase handling, not to contaminate RNase-free areas. >> Immunodetection 1% Blocking reagent 1hour 1/1000 anti-dig-ap in 1% Blocking reagent 2~5 hours at room temp or overnight at 4C TNT (TS7.5, 0.05% Tween20) 15 min x 3 TS min 1/50 NBT/BCIP in TS min~30 hours Block the sections with 1% Blocking reagent and incubate with anti-dig-ap. After washing in TNT (TS7.5, 0.05% Tween20), equilibrate the sections in TS9.5 for 10 min and develop color reactions with NBT/BCIP as a substrate. Monitor the color reactions using a binocular microscope from time to time. If the mrna is very abundant, you will start to see colors after 30min. Generally, it will take several hours to fully develop the colors. You have to decide when to stop the AP reactions by considering the desired signal-to-noise ratio. Depending on the type of tissue and how

10 it is fixed, you can continue AP reactions for over 24 hours without having too much background. For some tissue, you need to stop the reactions after several hours. >>mounting samples Stop reaction in PBS containing 10 mm EDTA. You can store the stained sections at 4C for a week or so. But it is probably safer to mount the sections as soon as possible. Mounting could be difficult, especially when you are using a harsh ProK condition for ISH. I mount the sections using two paint brushes with a section and a slideglass soaked in 1:1 solution of PBS and 0.1% gelatin, 40 % ethanol. After mounting, I dry the sections in a dark cool place for at least one week but this can be done overnight. Some people in my lab prefer it to be processed as soon as possible. After drying, I envelop the slides in a series of methanol/ethanol/xylen as follows. 100 % Methanol 10 min 100 % Ethanol 10 min x2 100% Xylen 10 min x2 Envelop in Entellan The most important thing here is to dehydrate the sample completely. So use fresh ethanol. If the dehydration is incomplete, NBT/BCIP reaction products may diffuse in Xylen. Treatment of sections first in methanol is my favorite protocol. This step somehow removes reddish color from the sections and the slide becomes cleaner blue. Removing unwanted background staining can be done in a series of % ethanol, and people usually do that.