BASIC DAB IMMUNOCYTOCHEMISTRY

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This protocol is for processing relatively thin sections (10-20 um) on slides (see A in NOTES below). Preferred method if enough antibody is available is to place slides in slide mailers and gently rock on rotating plate. An alternative is to lie the slides flat in a humidity chamber (Note B below) .

DAY 1

1) Equilibrate slides in buffer (PBS or PB)(Note C)

1) Hydrogen Peroxide treatment (to remove endogenous peroxidase from blood cells): 3% H2O2 in PBS (Note D)

2) Preblock (Note E): 1x PBS (pH 7.2) \"plus\" containing 0.5% BSA (Fraction V molecular grade only), 0.1% goat.0.1% triton-x-100
                    - 3 hours with gentle rocking

3) If endogenous biotin thought to be present – block with avidin-biotin (Note F)

4) Well-fixed or over fixed human tissue or for epitopes thought to be buried – use antigen retrieval method (Note G)

5) Primary antibody: Antibody of interest diluted into 1x PBS containing 0.5% BSA, 0.1% triton, and 0.1% filtered goat (Note H) serum (16-18 hr. - 4 C) (Note I)


DAY 2

1) Wash: 1x PBS with 0.5% BSA, 0.1% GS,0.1%triton   (5x 5 min. ea.)

2) Secondary Antibody: 1/1000 dilution (Note J) of biotinylated goat anti-mouse (Note K) (if secondary antibody was raised in goat and the primary was raised in mouse) in 1x PBS, 0.5% BSA, 0.1% GS,0.1% triton  (1.5 hrs., rt)

3) Wash: 1x PBS only (5x 5 min. ea.) (Note L)

4) Avidin: per ABC ELITE kit from Vectastain    (1 hr., rt)

Preferred method: 2ulA/ml + 2ul B/ml (Note M) (note that one ‘drop” of each is approx. 40 ul) – best to dispense drops into eppendorf tube and then use pipette for accurate measurement.

5) Wash: 1x PBS only               (2x 5 min. ea).

6) wash: 0.05 M TBS, pH 7.5           (3x 10min)
(DAB can precipitate in PBS, it is best to avoid the possibility - see Vector Labs handout on troubleshooting immunocytochemistry)

7) Chromagen: DAB per Vectastain kit (Note N)

(See Vectastain note on drops – ul conversions can be  determined. Mix each reagent well before adding the next. Prepare no more than 10-15 minutes ahead of time for best reaction).

2 drops buffer

4 drops DAB
2 drops hydrogen peroxide
2 drops nickel (if using)

10) Stop: H2O or TBS, pH 7.5 (NOT PBS!!)    5 min.

11) Final washes - 0.05M TBS, pH 7.6      3x, 5 min. ea.
If need to switch to PBS at this time you can, precipitation issues should be over

12) Hold in 1x PBS at 4o C overnight before mounting or continue with subsequent procedures (for example, counterstaining)

13) Dehydrate through alcohols (starting with 30%) and clear in xylene (Note O)





NOTES:

Note A. Regarding Standard optical requirements

The PMIC Core microscopes require the standard recommended coverslips and microscope slides for best visualization of approximately 0.17 thickness.

Coverslips – all objectives in Core require coverslips that average 0.17 thickness. Examples known to work: VWR 1.5 coverslips.

Microscope slides – the Core can accommodate 1 x 3 or larger slides – but it is imperative that the thickness of the slide be standard for research microscopes.  Examples known to work: Superfrost Slides (VWR).

Viscosity of Mounting Media – especially if high magnification work (100x) is to be performed, the low viscosity mounting media is recommended. Example known to work: VWR Low Viscosity Mounting Media.


Note B. About humidity chambers

The purpose of the humidity chambers is to provide a moist environment so that antibody placed on slides lying flat does not evaporate. Two commonly used chambers are: 1) Rubbermaid rectangular boxes with cooking racks placed inside (make sure the racks are level). 2) Western blot gel chambers (large square plastic boxes) with thin 2 ml pipettes cut to fit in parallel rows within the chamber. Tape the slides on the bottom of the chamber.

For both chambers – distilled water is added to just cover the paper towels sitting on the bottom of the chambers – do not want too much water. Place lid on boxes during all incubations.

Note C. About choice of buffer

PBS or Phosphate buffer are commonly used for immunocytochemistry. Which buffer is chosen depends on the investigators choice to some extent – but it should be noted that PBS can react and form a precipitate with DAB (see original Vector Lab troubleshooting information for DAB). Therefore, if PBS is the buffer used – it is best to wash in a tris based buffer prior to the DAB reaction.  

1x PBS freshly prepared or 0.15M Phosphate Buffer freshly prepared are recommended.


Note D. Regarding Hydrogen Peroxide Treatment

i) Use freshly made PBS – this is important since bacteria grows in PBS sitting in carboys for long periods of time which will interfere with the enzyme reactions occurring during the ICC procedure (Vector Lab Notes give more information).

ii) Use fresh or well sealed hydrogen peroxide. The addition of the hydrogen peroxide is crucial for the DAB reaction to proceed well. Less than optimal hydrogen peroxide will result in less than optimal DAB reactions.

Note E. Regarding Endogenous Biotin is visible in sections blocked for long periods of time but faint staining of some of the cells is still visualized. Use of an avidin-biotin Block will reduce the nonspecific background staining.  Preferred method: Vector Labs for A-B background removal. 15 minutes Avidin, wash in PBS, 15 minutes Biotin.

Note F. Antigen Retrieval Methods

There are a number of antigen retrieval methods. My preference is a sodium citrate bath heated in a microwave oven. This is a retrieval method used as a standard in pathology laboratories for antigen retrieval. Commercial antigen retrieval methods are available. How well they perform depends on their ability to uncover the epitope of your specific antigen. You may need to try a few antigen retrieval methods.

Note G. Notes Regarding Preblock

i) If you buy “bulk” serum (for example a 500 ml bottle of goat serum) the serum will needed to be filtered through a 0.2 um filter – either bottle top (if filter all at once) or syringe (if filter only needed aliquots.) Failure to filter will result in large “clumps” of debris over the section.

ii) BSA – best to purchase molecular grade Fraction V only (especially if combining with other procedures

Note H. Notes Regarding Serum

The serum is used to block nonspecific binding of the secondary antibody. Therefore, if you are using a goat anti-mouse secondary antibody, the serum you would block with is goat serum (filtered). Serums are available for all species used to make secondary antibodies. Goat is one of the most common secondary antibody species, but horse and donkey are also routinely used.

Note I. Primary antibody information

Some antibodies need signicantly longer primary antibody incubations. For example, anti-tyrosine hydroxylase requires at least a 48 hour incubation at 4C, AT8 also requires a 48 hour inc. Note that higher concentrations of these antibodies tends to raise the backgrounds – hence using a lower concentration for a longer period of time is best).

Note J. Secondary antibody information

The choice of the secondary should be carefully considered. Secondary antibodies with minimal cross-reactivity to the species your tissue is derived from will result in substantially lower backgrounds. If you are multiple labeling (two different peroxidase colorimetric reactions) using a secondary specific for multiple labeling is best. See Vector Labs extensive multiple labeling notes or Jackson Immunoresearch multiple labeling notes for more information.

Note K.  Dilution of the secondary antibody

For a majority of antibodies the secondary antibody dilution and the concentrations of all subsequent reactions should remain standard. If too high a background is obtained, troubleshooting the antibody in a dilution series is the best method for obtaining strong signal-to-noise ratios. If the antibody is particularly scarce the secondary antibody concentrations may need to be troubleshot.

Also, obtaining primary antibodies that have been stringently prepared and rigourously tested (this is especially true of commercial antibodies) is highly recommended. What you save in money by choosing an antibody of lesser price may also be an unwitting choice of a poorer quality antibody due to the preparation methods used. How well the primary antibody is prepared is ultimately what will determine the outcome of the signal to noise ratio.


Note L. About the wash after the secondary antibody incubation

i) IMPORTANT: make sure no sources of biotin are in the solutions that streptavidin will come in contact with - otherwise you will have a reduction in sensitivity due to non-specific biotin-streptavidin solution reactions

ii) Serum is a definite source of biotin – washes need to be performed thoroughly to remove all non-bound serum from sections.


Note M. Method troubleshot by John Olschowka et al.


Note N. DAB – even from the kit – is VERY TOXIC – be very careful in handling, do not inhale vapors, etc (see MSDS SHEETS). The kit makes it easier to use DAB, but the DAB is still a known carcinogen!!!!!, even in liquid form (it is significantly more hazardous in the powder form which is why it is not sold that way anymore apparently). Dispose of through hazardous waste, do not deactivate with chlorox. See notes from Mike M.


Note O. About the choice of clearing reagent

Although reagents such as “HISTOCLEAR” are less toxic than xylene (xylene is a known neurotoxicant and needs to be used in a fume hood), the “clarification” of the section using xylene is superior. This is a debatable point for some, not for others. At any rate, for best results, using fresh xylene to clear the section before mounting is recommended.