TITLE: In situ PCR using leaf epidermal peels
AUTHORS: Diep R. Ganguly, Asmini Athman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Method for visualizing cell-localized gene expression patterns.</div><div class = "text-block">Adapted from: </div><div class = "text-block">Athman, A., Tanz, S.K., Conn, V.M., Jordans, C., Mayo, G.M., Ng, W.W., Burton, R.A., Conn, S.J., and Gilliham, M. (2014). Protocol: a fast and simple in situ PCR method for localising gene expression in plant tissue. Plant Methods 10: 29.</div><div class = "text-block">Also, for more information on epidermal peels: </div><div class = "text-block">Chen, Z.-H., Eisenach, C., Xu, X.-Q., Hills, A., and Blatt, M.R. (2012). Protocol: optimised electrophyiological analysis of intact guard cells from Arabidopsis. Plant Methods 8: 15.</div></div>

[STEPS]
?. [Slide preparation (day before)]
The day before: prepare slides by placing frame seals on glass slide and apply pressure to borders to seal. These can be heated in thermal cycler at 90 °C for 10 minutes. Cut off excess seal to allow slide to sit appropriately in PCR machine.
?. [Sample preparation and fixation]
Prepare fresh fixative and keep on ice:Prepare fresh and use within 3 hours.
?. [Washes]
?. [DNAse treatment]
Setup Turbo DNAse (Ambion) mastermix.  AB1Component
x1 rxn (μl)2sterile milliQ H2087
310X Buffer
10
4DNase
3
AB1Component
x1 rxn (μl)2sterile milliQ H2087
310X Buffer
10
4DNase
3
?. [Reverse transcription]
Setup RT mix (Thermoscript RT or Superscript III)  AB1Componentx1 rxn (μl)2Sterile milliQ H206135X Buffer20410 mM dNTPs105Reverse primer (20 uM)2.56DTT 0.1 M5
AB1Componentx1 rxn (μl)2Sterile milliQ H206135X Buffer20410 mM dNTPs105Reverse primer (20 uM)2.56DTT 0.1 M5
?. [Sample preparation and fixation]
Bring plants to fume hood for epidermal peeling along with fixative, microscope slide and frame-seal. Keep all on ice for fixing and washing peels.
?. [Sample preparation and fixation]
Add 100ul fixative onto all microscope slides.
?. [Sample preparation and fixation]
Make epidermal peels (try to place 3-5 moderately sized peels per slide) and place directly into fixative on slide using a fine paint brush. Randomise which peels go to which slide (at this point, there should be no allocation of which slide will be used for which gene – can make this decision based on peel + slide quality). Wear gloves, handle formaldehyde with care.
?. [Sample preparation and fixation]
Incubate peels in fixative for 45 minutes, refreshing the fixative (with pipette) approximately every 15 minutes (apply evenly across peels on each slide).
?. [Washes]
From this point, the remainder of the protocol can be performed at lab bench. Wipe off any excess liquid to ensure no formaldehyde remains.SAFE PAUSE POINT
?. [Washes]
Wash 3 x 5 min in 1x PBS (0.1 M Na2HPO4, 1.3 M NaCl, pH 7.5, stored at room temperature
?. [Washes]
After last wash, remove as much wash buffer as possible.
?. [DNAse treatment]
Heat inactivate at 70 °C for 15 mins. Place on ice.NOTE: Do not cool in thermal cycler as it can lead to cracking of glass slides.
?. [DNAse treatment]
Add 3 μl 0.5 M EDTA to give final concentration of 15 mM.
?. [DNAse treatment]
Remove PBS solution and add 100 μl of mastermix to each slide.
?. [DNAse treatment]
Incubate 37 °C for 45 mins.
?. [DNAse treatment]
At this point, it must be decided which genes are being studied on which slides. There must be two control slides: one positive housekeeper (e.g. 18S rRNA) control and one no RT housekeeper negative control.
?. [DNAse treatment]
Remove DNase mix and wash 2x with ice-cold sterilised water. Do not remove final wash before RT reaction mix is ready!
?. [Reverse transcription]
Add 98.5 µl of mastermix to all slides
?. [Reverse transcription]
Incubate slides at 65 °C for 5 minutes, then place on ice for 1 min.
?. [Reverse transcription]
Add 1.5 µl of RT enzyme (Thermoscript or Superscript III) to all slides EXCEPT negative housekeeper control. Pipette up-and-down gently to mix around.
?. [Reverse transcription]
Run thermal cycle at 55 °C for 30 mins, 70 °C for 15 mins, then hold at 4°C (better to take out of machine and keep on ice). This is a safe PAUSE POINT (keep slides covered in fridge).
?. [In situ PCR]
Remove RT solution from slides (keep slides on ice).
?. [In situ PCR]
Wash slides twice for 1 min with 100 µl ice-cold sterile water. When ready to proceed, remove as much water as possible (use a 10/20 µl pipette to get as much as possible).
?. [In situ PCR]
Setup PCR mix (HiFi Phusion Taq)  AB1Componentper slide (µl)2Sterile milliQ H2074.635X Buffer20410 mM dNTPs25polymerase0.56DIG-11-dUTP (1 mM)0.47Reverse primer (20 µM)1.258Forward primer (20 µM)1.25
AB1Componentper slide (µl)2Sterile milliQ H2074.635X Buffer20410 mM dNTPs25polymerase0.56DIG-11-dUTP (1 mM)0.47Reverse primer (20 µM)1.258Forward primer (20 µM)1.25
?. [In situ PCR]
Distribute PCR mix equally across the slide by pipetting and run PCR:95 °C 30 min95 °C 10 min60 °C 25 min72 °C 5 minReturn to step 2 20–35x72 °C 7 minutes10 °C hold (better to take slide out).
?. [Colorimetric detection of DIG labelled products]
Remove frame-seal chamber completely and perform remainder directly on slide. During incubation, place slide in small plastic box to minimise evaporation.
?. [Colorimetric detection of DIG labelled products]
Pipette off remaining PCR solution.
?. [Colorimetric detection of DIG labelled products]
Wash twice for 5 min in 100 µl 1x PBS.
?. [Colorimetric detection of DIG labelled products]
Gently add 100 µl of 1x Block solution and incubate 30 min on ice.10X Block solution: 10 mg BSA in 1 mL 10x PBS
?. [Colorimetric detection of DIG labelled products]
Dilute anti-DIG-AP antibody 1:500 in 1x Block solution, keep on ice.
?. [Colorimetric detection of DIG labelled products]
Pipette off as much 1X block solution as possible.
?. [Colorimetric detection of DIG labelled products]
Add 50 µl of diluted anti-DIG-AP antibody and incubate at room temperature for 1 hour.
?. [Colorimetric detection of DIG labelled products]
Remove antibody solution and wash twice for 15 min at room temperature in Wash Buffer 2 (can transfer peels to another microscope slide if desired; keep in wash buffer 2).Wash Buffer 2: 0.1 M TRIS-HCl, 0.15 M NaCl, pH 9.5
?. [Colorimetric detection of DIG labelled products]
Remove wash buffer 2.
?. [Colorimetric detection of DIG labelled products]
Add 50 µl BM purple (AP substrate). This is light sensitive, incubate in dark (e.g. drawer, or cover) and keep on ice.
?. [Colorimetric detection of DIG labelled products]
Staining can occur anywhere between 10 min – 2 hours. Optimize as appropriate. If no signal is detected after 1 hour, refresh BM purple (take off and add another 50 µl).
?. [Microscopy]
Once stain has developed, pipette off BM purple.
?. [Microscopy]
Add 100 µl Wash Buffer 2 and wash 3x 5 min to remove residual stain and debris.
?. [Microscopy]
Wash once with sterile water, then remove all water.
?. [Microscopy]
Mount section in 40 µl 40% glycerol (40–150 µl depending on number of peels on slide). Do not use excessive amounts of glycerol.
?. [Microscopy]
Place coverslip on top and seal corners with nail varnish. Coverslip should not float (remove some glycerol if needed). NB do not slide coverslip, instead just drop directly onto peels/glycerol.
?. [Microscopy]
Visualise under bright field microscope. Slides can be stored at room temp and is good ~3 weeks. Ensure all peels are imaged using the same settings.