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 H₂ O 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

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