Immunohistochemistry for VHL on mouse tissue with M.O.M kit (Catalog No. PK-2200)
Before starting:
§ First day
· Prepare 1000 mL TBS
o 100 mL TBS 10x
o 900 mL H2O
· Preheat the waterbath and the citrate solution to 95° C
· Prepare the humidified chamber with TBS
· Thaw TNB
§ Second day
· Prepare 2L TBS
o 200 mL TBS
o 1800 mL H2O
- 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 start until the Citrate solution reaches 95°C!!!
- Deparaffinize as usual:
o 3 times in xylene for 5 minutes
o 3 times in EtOH for 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 3% 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 (Invitrogen 00-8877) and place in a TBS humidified chamber.
- Incubate sections for 1 hour in working solution of M.O.M.™ Mouse Ig Blocking Reagent prepared as described in the kit.
- Wash sections 2 x 2 minutes in TBS.
- Incubate tissue sections for 5 minutes in working solution of M.O.M.™ diluent prepared as described in the kit.
- Tip excess of M.O.M.™ diluent off sections. Dilute primary antibody in M.O.M.™ diluent (Primary antibody: BD 556347 mouse anti-VHL dilution 1:50. The negative control slides get only M.O.M.™ diluent). Incubate overnight in cold room. Remember to choose a negative control per each slide that will not get the antibody.
~~~~~~~~~~~~~~~~~~~~~~~ The next day ~~~~~~~~~~~~~~~~~~~~~~~
Let the slides at room temperature for 30 mins.
- Wash sections for 2 x 2 minutes in 1X TBS.
- Apply working solution of M.O.M.™ Biotinylated Anti-Mouse IgG Reagent prepared as described in the kit. Incubate sections for 30 minutes at RT.
- Wash sections for 2 x 2 minutes in TBS.
- Apply VECTASTAIN® ABC Reagent prepared as described in the kit. Incubate sections for 5 minutes.
- Wash sections for 2 x 5 minutes in TBS.
- 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 5 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 TLW on 05/03/10