Immunostaining-Monoclonal (Mouse) antibodies



Immunostaining-DAKO Envision Plus Kits

Notes:

For each tissue array, 200-400ul of each incubating solution is needed.

If slides are not baked, bake for at least one hour at 60C making sure oven does not go over 60C

Procedure is done at room temperature except for microwaving steps.

Use DAKO Antibody Diluent, Background Reducing (cat# S3022) to dilute antibodies.

DAKO HRP secondary antibodies Mouse cat# K4000 or K4001, Rabbit cat# K4002 or K4003

Staining procedure

Deparaffinize slides through three changes of xylene, incubating slides10 min in each change.

Hydrate slides to water by dipping them twenty times in each of three changes of 100% ethanol, two changes of 95% ethanol, one of 70 % ethanol, and four changes of distilled water.

Place slides in microwave slide dish leaving a blank space between each slide (plastic works best with a plastic slide rack) and cover with antigen retrieval buffer (we use citrate pH6 or EDTA pH8 in most cases. Recipes follow.).

Microwave on high power for 3 minutes if one rack of slides, 8 minutes if two racks of slides and 13 minutes if three racks of slides.

Continue microwaving without opening or stopping microwave at 80% power for 12 minutes. (Times may vary by microwave, but the important thing is to make sure the slides boil rapidly for 10 minutes minimum and do not dry out. An additional container of buffer may be microwaved together with the slides to allow for refilling of the slide dishes.)

Cool for a minimum of 30 minutes. Cooling time is critical.

Rinse in distilled water, X4.

Block endogenous peroxidase activity by covering tissue with 1% aqeous H2O2 and incubating for 10 minutes.

Rinse in PBS, X2

Cover tissue with DAKO Serum-Free Protein Block (cat# X0909) and incubate for 20 minutes.

Wipe off excess block and immediately cover tissue with primary antibody or negative control solution and incubate for 30 minutes.

Rinse in PBS, X2

Cover tissue with appropriate DAKO HRP secondary and incubate for 30 minutes.

Rinse in PBS, X2

Just before use, prepare DAKO DAB (cat# K3468) according to directions.

Cover tissue with prepared DAB and incubate 10 minutes. Keeping this time constant allows for more accurate duplication of titers.

Rinse with distilled water, X5

Counterstain with mayer’s hematoxylin (DAKO cat# S3309) for 1 minute.

Rinse with tap water, X3.

Dip slides three times in dilute ammonia water or lithium carbonate water (a slightly basic solution is all that is needed) to blue.

Rinse in tap water, X5.

Dehydrate slides by dipping them twenty times in one change of 70% ethanol, two changes of 95% ethanol and three changes of 100% ethanol.

Clear slides by dipping twenty times in three changes of xylene.

Coverslip slides with Protex or other xylene-compatible mounting medium.

Solutions:

10X 10mM Citrate Buffer pH 6 (2 liters)

Measure 2 liter distilled water

Add 42gm of citrate acid monohydrate (fw=210.1)

Adjust pH to 6.0 with sodium hydroxide or HCl as necessary

1X 10mM citrate pH6

10X 10mM Citrate pH6 ……..100ml

Millipore H2O………………..900ml

10X 1mM EDTA pH8 (2 liters)

EDTA……………………………7.4 g (fw=372.24)

Millipore water………………..2000.0mL

1X 1mM EDTA pH8

10X 1mM EDTA pH8………..100ml

Millipore H2O………………..900ml

25X PBS (phosphate buffer – 2 liters)

360g Sodium Chloride

66g Sodium phosphate (monobasic, monohydrate)

376g Potassium phosphate (dibasic)

add distilled water up to 2 liters. (pH should be around 7.3)

1X PBS (working solution)

25X PBS stock solution…………40.0mL

Millipore water………………...960.0mL

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