HRP STAINING:      - Mix DAB solution as indicated in recipe.

                        - Immerse slides in DAB solution until colour develops

  sufficiently (maximum of 20 min.)

                                    - Rinse thoroughly  with 0.01 M PBS.

Phosphate Buffered Saline (PBS)

Working Solution (0.01M or 1x)              Stock (0.1M or 10x)

NaCl                                       8.0 g  (137 mM)                                             80.0 g

KCl                                          0.2 g  (2.7 mM)                                              2.0 g

Na₂HPO₄.7H₂O                     2.68 g  (10 mM)                                             26.8 g

KH₂PO₄                                  0.24 g  (1.76 mM)                                         2.4 g

-  Dissolve in 800 mL dH₂O.

-  pH to 7.4.

-  Q.S. to 1000 mL with dH₂O.

-  Autoclave (optional for working solution if it will be used within a couple of days).

Working Solution (0.01M or 1x):   Dilute Stock 1/10 with dH₂O.

1^o Ab Diluent:           9 mL   0.01 M PBS

 (10 mL)                      1 mL   Horse Serum (10%)

10 uL  10% Na azide (0.01%); Do not use for HRP reactions.

0.1 g   Bovine Serum Albumin  (1%)

2^o Ab Diluent:           0.1 g   Bovine Serum Albumin (1%)

  (10 mL)                     10 mL 0.01 M PBS

Heat Inactivation of Serum

  It is advisable to heat treat serum used in Ab diluents; this will decrease

  background staining.

·          Pour serum into small glass bottles (125 mL), cap loosely and put in

65^o C waterbath for 30 min.  Cool serum to room temp.  and filter sterilize.

Aliquot into 0.5 mL and 1 mL volumes.  Store at - 20^o C.

1 uM Bis Benzimide

·          1 uM Bis Benzimide is equivalent to a concentration of 0.534 mg/L

·          Dissolve Bis Benzimide in an appropriate volume of sterile 1x PBS.

·          Cover bottle with aluminum foil and store at 4^o C.

Staining:   Immerse slides in 1 uM Bis Benzimide for 30 sec. then rinse with

1x PBS.  Slides need to be rinsed thoroughly so treat with bis benzimide early

in the procedure in order to take advantage of subsequent rinsing (e.g. stain during

rinsing after Primary Ab).

·          Look at the slides under the fluorescent microscope using the blue cube.

Ethidium Bromide

·          Add 4 uL of Ethidium Bromide Stock (10 mg/mL) to 50 mL 1x PBS

(final concentration will be 0.00008 % or  0.8 ug/mL).

Staining:   Immerse slides in working solution for 10 min.  then rinse with 1x PBS.

Slides need to be rinsed  thoroughly so treat with ethidium bromide early in the

procedure in order to take advantage of subsequent rinsing (e.g. stain during rinsing

after Primary Ab).

·          Look at the slides under the fluorescent microscope using the red cube.

N.B.   Do not stain nuclei with ethidium bromide if you will be counterstaining

sections with 1% Fast Green FCF.

The Ethidium Bromide Working Solution will need to be decontaminated.

Add 100 mg of activated charcoal for each 100 mL of solution. Let sit at R.T.

for at least 1 hr.,  swirling  solution from time to time.  Filter solution

and discard liquid.  Dispose of charcoal waste through safety office.

Controls:  Gut sections -    1 negative control

       -    1 positive control  (not necessary if one of the slides

             being stained contains gut)

1^o Ab Diluent (10 mL):        9 mL 0.01 M PBS

1 mL horse serum (10%)

0.1 g Bovine Serum Albumin  (1%)

2^o Ab Diluent (10 mL):        0.1 g Bovine Serum Albumin (1%)

                                                10 mL 0.01 M PBS

Acid Alcohol

To make:                                          50 mL100 mL          350 mL

Absolute Alcohol (90%)                    45 mL90 mL315 mL

ddH₂O                                                2.5 mL            5 mL               17.5 mL

Glacial Acetic Acid (5%)                  2.5 mL            5 mL               17.5 mL

* Mix ingredients in the order given.  Prepare and use in fumehood.

DAB Working Solution

1.                  Dissolve  1 mL DAB stock solution (stock conc. 10 - 25 mg/ mL)  in

50 mL Phosphate Buffer or 1x PBS.

2.                  Add 100 uL of Colour Intensifier; add slowly, stir continuously.

3.                  While stirring, add fairly fresh H₂O₂.  Use Immediately,  solution is

good for only 20 min.

Add:   3%  H₂O₂       

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If using   10 mg DAB                        100 uL                       

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If using   25 mg DAB                        200 uL                       

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N.B.  DAB is carcinogenic; take the necessary precautions.

DAB Disposal         

Pour some bleach into a large container and to this add the used DAB

solution and the dH₂O from the first two rinses.   After letting it sit for

at least 10 min., this mixture may be poured down the drain followed

by copious amounts of water.  Please note that a large volume of bleach

is  not necessary; after adding the DAB and the water from the first two

rinses, you should end up with a mixture that is about 10-20 % bleach.

Rinse anything that may have come in contact with the DAB solution

with a dilute bleach solution (10-20%) and then thoroughly with water.

Phosphate Buffer (0.05 M)

Na₂HPO₄                    5.75 g

NaH₂PO₄.2H₂O         1.48 g

·          Dissolve in 1 L dH₂O.

Colour Intensifier

NiCl₂.6H₂O*   (5%)                0.5 g

CoCl₂.6H₂O*  (5%)                0.5 g

- Dissolve in 10 mL ddH₂O. Transfer to a sterile vial.  Wrap vial with aluminum

foil; store at 4^o C.

-Add 100 uL to 50 mL DAB Working Solution (use a syringe to withdraw

colour intensifier from vial).

N.B.  Either of the above metal ions may be used alone if its concentration

is increased to 10%.  Cobalt is preferable since the colour produced is more

stable.

* Toxic - wear gloves, take necessary precautions.

DAB Stock Solution

1 g  DAB Powder  (Sigma D5637)

·          Add 10 mL 1x PBS to DAB container.  Put on cap and invert several

times to mix.  Let sit for about 10 min.

·          Transfer DAB solution to beaker.  Rinse DAB container with 10 mL

1x PBS, and add  to beaker.  Add an additional  20 mL 1x PBS to beaker

(Total volume should be 40 mL).

·          Mix well.  Filter DAB Solution through a syringe filter.

·          Aliquot into 1 mL volumes.  Concentration of aliquots is 25 mg/mL.

Store at -20^o C.

N.B.  DAB is a powerful carcinogen.  Exercise due caution.  Prepare

stock solution in fumehood; wear gloves and face mask.  Clean up

thoroughly and rinse everything that has come into contact with

the DAB solution with a 20% bleach solution.