Section in situ hybridization (SISH) on kidney sections (Version 1.0)

Prepare fresh 4% PFA 

- 200 ml/big jar	40 ml/small jar
- Xylene 1 		10 min
- Xylene 2 		10 min
- 100% EtOH 		5 min
- 95% EtOH 		5 min
- 80% EtOH		5 min
- 60% EtOH 		5 min
- 30% EtOH		5 min
- PBS 			5 min
- fresh 4% PFA/PBS	10 min		(do not discard!)
- PBS 			5 min
- PBS			5 min

- 10 µg/ml Proteinase K/PBS (Prot. K stock 1,000x)		 
    Mouse embryonic kidney and human 8-9 weeks frozen section      20 min  
    Mouse adult tissue and human 13-17 weeks frozen section        40 min 
- 15 µg/ml Proteinase K/PBS (Prot. K stock 1,000x)
    Human 13-17 weeks  paraffin section				   40 min
- PBS 		        3x 3 min
- 4% PFA/PBS 	        5 min
- PBS 		        3x 3 min
- fresh Acetylation Solution		1x 10 min on stir plate!
- PBS 			3x 5 min
- 0.85% NaCl		3 min 
- 70% EtOH/0.85%NaCl	5 min
- 95% EtOH  		5 min
- *Little group: Incubate twice with pre-hyb buffer (GUDMAP recipe, pre-heated 65°C)
  *At this time the slides may incubate for as long as necessary until the chamber rack is at temperature and the probes are ready, generally between 10-20 min.
- McMahon: Air dry sections for 10 min – 2 hours

- 5x SSC pH4.5 (big jar) 				1x 5 min at 64°C
- 50% formamide/1xSSC pH4.5				3x 10 min at 64°C
- TNE 							2x 5 min at 37°C
- RNase A (2 µg/ml) in TNE (stock conc. 10 mg/ml)	15  min at 37°C
- TNE 							2x 5 min at 37°C
- 2x SSC pH4.5						2x 10 min at 64°C
- 0.2x SSC pH4.5					4x 10 min at 64°C

- 1x MBST				3x 5 min at RT
  circle around tissue with PapPen
- Blocking Buffer			1x 1h at RT 
- Antibody Solution 			overnight      (Yu) at 4°C
-  apply ~200 µl/slide and cover with parafilm, H2O-humidified chamber

- 1x MBST 					3x 5 min at RT
- NTMT pH9.5 + 2 mM Levamisole 			2x 5 min at RT
  Spin down BM purple 5 min at 2,000 rpm
- aliquot 200 µl of BM Purple directly onto slide
- incubate at RT in dark (fresh BM Purple every day)

- PBS  				1x 5 min  (--> alternatively, proceed with IHC)
- 4% PFA/PBS 			1x 10 min
- PBS  				2x 5 min
- rinse briefly in 70% EtOH, air-dry sections
- mount and coverslip with aqueous mounting media (Glycergel, 60°C)

- Xylene
- 100% EtOH
- 95% EtOH		285 ml EtOH + 15 ml H2O
- 80% EtOH 		240 ml EtOH + 60 ml H2O
- 60% EtOH		180 ml EtOH + 120 ml H2O
- 30% EtOH 		90 ml EtOH + 210 ml H2O
- 4% PFA/PBS		make fresh!
- Proteinase K (10 µg/ml) in PBS (stock conc. 10 mg/ml)
- Acetylation Solution
  - H2O					200 ml
  - triethanolamine			2.66 ml
  - 37% HCl				0.35 ml
  - begin stirring and add acetic anhydride   0.75 ml
- 20x SSC pH 4.5 (Sigma S-8015)
  - citric acid to pH 4.5; ~1.35 g/100 ml
  - 2x SSC        	20 ml 20x SSC, 180 ml H2O
  - 1x SSC        	10 ml 20x SSC, 190 ml H2O
  - 0.5x SSC      	 5 ml 20x SSC, 195 ml H2O
  - 0.2x SSC      	 2 ml 20x SSC, 198 ml H2O
- pre-hyb solution:	formamide 	25 ml	(50%)
  (deionized! Sigma F9037)
  (McMahon)   5x SSC pH 4.5		12.5 ml 20x	(5x)
	      1% SDS			 2.5 ml 20%	(1%)
	      yeast tRNA		100 µl		(50 µg/ml)
	      (Invitrogen 15401-011) (25 mg/ml)
	      heparin 			250 µl		(50 µg/ml) 
	      (Sigma H3393-500KU) (10 mg/ml)
	      H2O			9.65 ml	

- Hybridization Buffer (Little group protocol)
    formamide	100%			25 ml		(50%)
    20x SSC pH 4.5 			 5 ml 		(2x)
    50x Denhardt's			 1 ml		(1x)
    50% dextran sulfate			10 ml		(10%)
    10 mg/ml tRNA (50x)			 1 ml		(0.2 mg/ml)
    25 mg/ml ssDNA (50x)		 1 ml		(0.5 mg/ml)
    H2O				     to 50 ml
- 50% dextran sulfate stock
  Dissolve 50 g dextran sulfate in 100 ml H2O. 
  Add powder a little at a time to the water on a heated magnetic stirrer to avoid clumping. 
  Once dissolved, aliquot and store at -20°C.
- 50x tRNA stock
  Dissolve 200 mg in 20 ml H2O and rotate in hyb oven at 55-65°C o/n.
  Aliquot into 1 ml tubes and store at -20°C.
- 50x salmon sperm DNA stock
  Mix 500 mg in 10 ml H2O, shear with a 17 gauge needle and adjust volume to 20 ml.
  Aliquot into 1 ml tubes and store at -20°C.

- Stringency Wash Buffer 1 (50% formamide, 1x SSC)
  250 ml formamide + 25 ml 20x SSC+ 225 ml H2O
- TNE (10 mM Tris pH7.5, 500 mM NaCl, 1 mM EDTA)
    1M Tris pH7.5		      	 10 ml
    5M NaCl			      	100 ml
    0.5M EDTA			      	  2 ml
    H2O				      	ad 1L
- 10x MBS stock			
    116.07 g maleic acid (Sigma M0375) 			 (1M)
    (exothermic! starts clearing around correct pH)
    5M NaCl			      	300 ml		 (1.5M) 
    pH7.5 with NaOH 		      	(start with 70 g/l)
    H2O				      	ad 1l 	
-1x MBST
    dilute from 10x with H2O
    add Tween-20 to 0.1%

- 2% BR/MBST Blocking Reagent (Roche 11096176001) 
  2 g of blocking reagent in 100 ml 1x MBST
  Place on heat stirrer at 65°C for at least 60 min
- Blocking Buffer (20% HISS, 2% BR/MBST)
    10 ml heat-inactivated sheep serum
    40 ml 2% BR/MBST
- Antibody Solution 
    (anti-DIG Fab (Roche 11093274910) 1:4,000 in 2% BR/MBST/1% HISS )
    anti-DIG (1G8)	 	      12.5 µl 
    2% BR/MBST 			      49.5 ml 
    HISS (1%)			       0.5 ml 
- NTMT (0.1M NaCl, 0.1M Tris pH9.5, 50 mM MgCl2, 0.1% Tween-20 w/v)
    1M MgCl2		     	       5 ml
    10% Tween-20		       1 ml
    5M NaCl			       2 ml
    1M Tris pH9.5 		      10 ml
    H2O				      82 ml

    + 2mM Levamisole (Sigma L9756-10g – Tetramisol hydrochloride)
    240.76 MW x 0.002 M x 0.050 l
    24 mg in 50 ml NTML. Prepare in fumehood.