ImmunoHistoChemistry

For Cadherin 11 on Formalin Fixed Paraffin Embedded Tissues

Before starting:

§ First day

· Prepare 500 mL TBS

o 50 mL TBS 10x

o 450 mL dH2O

· Preheat the waterbath and the citrate solution to 95° C

· Prepare the humidified chamber with TBS

· Thaw TNB (from the TSA kit)

§ Second day

· Prepare 4L TNT

o 400 mL TBS

o 3600 mL dH2O

o 2 mL Tween-20 (add while stirring)

Protocol using the TSA kit (Perkin Elmer #NEL700A):

- Let slides dry overnight on slide warmer at 60°C. Remember to pick also a negative control per each slide that will not be incubated with the primary antibody.

- Do not star until the Citrate solution reaches 95°C!!!

- Deparaffinize as usual:

o 3 times in xylene 5 minutes

o 2 times in 100% EtOH 1 minute

o 1 times in 95% EtOH 1 minute

- Wash in TBS for 5 minutes

ANTIGEN RETRIEVAL

o SODIUM CITRATE

§ 12.5 ml Citrate Buffer pH 6.0 (Invitrogen 00-5000)

§ 237.5 ml ddH2O

§ Microwave buffer to about 90 – 95° C (2.5 min)

§ Place it into preheated waterbath 95° C and wait until temperature is reached.

§ Put slides in buffer for 10 mins at 95° C

§ Let solution and slides cool to RT for about 30 mins

- Quench in 30% H2O2/MeOH, 10 min (25 ml 30% H2O2, 225 ml MeOH)

- Rinse in dH2O for 5 mins

- Rinse 1x in TBS (1 or 2 minutes)

- Dry the area around the sample. Circle with PAP PEN and place in a PBS humidified chamber.

- Incubate with TNB (~ 100mL per slide) at RT in moist chamber for 30 mins.

- Pour off TNB and add primary antibody diluted in TNB. Incubate overnight in the cold room. Remember to choose a negative control per each slide that will not get the antibody.

o Primary antibody:

Rabbit anti-Cadherin 11 Polyclonal (Invitrogen: 71-7600)

Diluted 1:100 in TNB

The negative control slides get only TNB.

~~~~~~~~~~~~~~~~~~~~~~~ The next day ~~~~~~~~~~~~~~~~~~~~~~~

Let the slides at room temperature for 30 mins and prepare TNT.

- Wash: 1X TNT, 3x 5mins at RT (on rocking platform)

- Add biotinylated secondary antibody diluted in TNB. Incubate for 30 mins at RT.

o Secondary antibody:

§ Dako Cytomation, Polyclonal Swine anti-Rabbit F(ab) IG biotinylated (E 0431) 1:300 in TNB

- Wash: 1X TNT, 3x 5mins at RT (on rocking platform)

- Add SA-HRP (from TSA kit) diluted 1:100 in TNB, in moist chamber for 30 min

- Wash: 1X TNT, 3x 5mins at RT (on rocking platform)

- Biotinyl Tyramide (from TSA kit), diluted in amplification diluent, 1:50, 5 mins RT in moist chamber.

- Wash: 1X TNT, 3x 5mins at RT (on rocking platform)

- Add SA-HRP (from TSA kit) diluted 1:100 in TNB, in moist chamber for 30 min

- Wash: 1X TNT, 3x 5min at RT (on rocking platform)

- For HRP detection, use DAB (Vector Peroxidase Substrate kit SK-4100)

Vector DAB

To 5 ml dH2O:

· add 2 drops buffer; mix well

· add 4 drops DAB; mix well

· add 2 drops H2O2mix well

- Incubate until color develops (monitor, but approx 3 mins)

- Wash 5 min in dH2O.

- Counterstain with Hematoxylin (Invitrogen 00-8001) for 3 min.

- Wash 5 min in cold tap water

- Incubate in 1X PBS 1 min

- Wash in dH2O

- Dehydrate (1min in EtOH for 3 times, 2 mins in Xylene for 3 times) and coverslip as usual

Updated by LM on 7/20/13