| |||||||
Protocol D.6
Dissociated Retina Immunohistochemistry
Solutions
100X trypsin
10 mg/ml in 1X PBS
Sigma #T2271
100X soybean trypsin inhibitor
10 mg/ml in 1X PBS
Sigma #T6522
1000X DNaseI
2mg/ml in HBSS
Sigma #D4513
Explant Culture Medium
45% HAMS F-12 (Gibco #11765-054)
45% DME (Gibco #10313-021)
10% FCS (HyClone)
Insulin (5 mg/ml 1000X stock in H2O with HCl)
(10 ml/liter) L-glutamine (200 mM, Cellgro # 25-005-CI)
(10 ml/liter) Penn/Strep (Cellgro #30-002-CI)
(10 ml/liter) HEPES (Cellgro #25-060-CI)
20% Paraformaldehyde/4% Paraformaldehyde-PBS
200 g paraformaldehyde
1 ml 10N NaOH
up to 1 liter with Q, heat to 65° to dissolve, aliquote and store at -20°
Mix 100 ml 20% Paraformaldehyde with 50 ml 10X PBS and bring up to 500 ml with Q
filter, and store at 4° for up to 2 weeks
1000X Poly-D Lysine
500 mg Poly-D Lysine (Sigma)
up to 50 ml with sterile PBS
store at -20° in 10 ml aliquotes
Block Solution
1X PBS
2 % normal serum (donkey, rabbit, goat)
0.5% Triton X-100
store at 4° C for 1-2 weeks
1000X DAPI
50 mg DAPI
up to 10 ml with 50% MeOH
store in a light proof bottle at -20° C for years
Other Reagents
biotinylated secondary antibodies (Vector labs Inc.)
ABC biotin/streptavidin (Vector labs Inc.)
tyramide reagent (see protocol T.4)
8 chamber slides (Cel-line/Eric Scientific Co., 1-800-258-0834, 12 mm 8 square slides, teflon coated)
Procedure
before starting see criteria/guidelines for dissociated cell scoring
• Wash the tissue (freshly dissected or explant) in prewarmed PBS (6 ml in a 6 cm dish).
• Transfer to an eppendorf tube with approximately 200 microliters PBS and add 20 microliters of trypsin stock.
• Incubate at 37° C for 5 minutes and during this incubation prepare the chamber slides by placing 1X poly-D lysine diluted in 1X PBS on the slides. After a 5 minute incubation wash twice with 1X PBS. Do not let them dry out.
• For tissue dissociation, titurate 3-5 times with a p1000 tip and return to the 37° water bath for 2-4 minutes. Titurate again to generate a single cell suspension.
• Add 20 microliters of soybean trypsin inhibitor, mix by inversion and add 2 microliters of DNaseI. Incubate at 37° for 5 minutes, titurate and add explant culture medium up to 1.5 ml.
• Transfer cells to chambe slides (105 cells per chamber is ideal) and incubate at 37° for 15-30 minutes in the tissue culture incubator.
• Aspirate the medium and immediately fix in 4% paraformaldehyde for 5 minutes.
• Wash twice in 1X PBS and incubate in 3% hydrogen peroxide in PBS for 5 minutes.
• Wash twice in PBS and block for 5-10 minutes.
• Add primary antibody (see Dyer and Cepko for dilutions) and incubate for 30 minutes.
• Wash 3 times in PBS and add secondary antibody at a dilution of 1:500.
• Incubate for 30 minutes, wash 3 times in PBS and add a few drops of ABC reagent.
• Wash 3 times in PBS, 1 time in TNB block (provided with tyramide kit) and perform tyramide amplification. For each slide mix 150 microliters of amplification diluent with 1 microliter of tyramide compound (NEN) or dilute the tyramide 1:50,000 if synthesized in house (see T.4).
• Incubate in tyramide for 10 minutes and stop by washing in PBS.
• Stain with DAPI for 5 minutes in PBS, wash 2 times in PBS and mount with gelvatol.