Protocol for immunohistochemical staining of PARAFFIN SECTIONS

Interpretable results from immunostained paraffin sections can be challenging if appropriate negative and positive controls and blocking agents are not employed.

Polyclonal antibodies usually produce good results on paraffin sections without a need for antigen retrieval methods.

Monoclonal antibodies may need antigen retrieval--and this may involve either Proteinase K or other enzyme treatment or the utilization different Heat Induced Antigen Retrieval (HIER) methods using either a citrate buffer at pH 6 or EDTA buffer pH 8, or other buffers.

One should plan to use multiple slides with positive and negative control tissues (or postiive and negative control cell pellets that have been processed, embedded and sectioned for use as paraffin section controls) and individually optimize the conditions for each antibody to be tested.

Detection using the biotinyl tyramide catalyzed signal amplification may also have to be used with certain monoclonal antibodies

The following protocol details the use of HIER for Antigen Retrieval (AR) to unmask epitopes , and then it uses biotinyl tyramide catalyzed signal amplification, which is useful to detect really low abundance epitopes

A. Tissue Controls:

1. Positive control: Cells or tissue expected to be positive. Positive controls may also be the cells that are know to express the epitope, that are grown in tissue culture up to about 100 million, taken and made into a pellet, fixed, paraffin embedded and sectioned.
2. Negative control: Cells or tissue expected to be negative.
3. Blocking peptide (see below)

B. Reagent controls:

1. 1% BSA/TBST reagent negative control for secondary reagents
2. Rabbit IgG Dako Cat.# 1505 Negative control reagent at 5 ug/ml
3. Rabbit anti human vWf Dako Cat# 0936 (pre-diluted). Positive for blood vessels in tissue.
4. Mouse anti Vimentin Dako Catalog# N1521 prediluted, ready to use, as a technical positive control when using mouse antibodies as test
5. Mouse IgG at 5 ug/ml
6. Further controls included staining of cells expressing higher levels of epitope in question, and using the immunizing peptide (1 mg/ml) at a 1:50 dilution to compete out the reactivity of the antibody, resulting in a negative stain.

C. Deparaffinization:

1. Label slides with date and reagent to be applied and take to the fume hood where the deparaffinization will occur
2. Xylene 1 - overnight OR for 10 minutes
3. Xylene 2-10 minutes, briefly move rack up and down (BMRUAD)-move slide holder up and down so that the slides get "washed" before leaving the slide rack in the tub of xylene for the specified amount of time
4. Xylene 3-10 minutes
5. Dip into 100% alcohol BMRUAD and then leave for 5 minutes, and repeat one more time
6. Dip into 95% alcohol BMRUAD and then leave for 5 minutes, and repeat one more time
7. Dip into 70% alcohol BMRUAD and then leave for 5 minutes, and repeat one more time
8. Dip into TBST buffer BMRUAD and then leave for 5 minutes, and repeat two more times

TBST= 50mM Tris/ 150mM NaCl/0.05% Tween 20

TBST for CSA 50mM Tris, 300 mM NaCl/ 0.05%Tween 20

D. Block ENDOGENOUS PEROXIDASES

1. Immerse slides in slide holder in 0.3% H2O2 (Paraffin sections) in TBST for 30 minutes at room temperature
2. follow this with 3 buffer washes.

E. Block ENDOGENOUS BIOTIN

1. Overlay with 0.1% avidin for 10 - 15 minutes (following manufacturer's suggestions)
2. followed by 3 buffer washes.
3. Overlay with 0.01% biotin for 10 - 15 minutes (following manufacturer's suggestions)
4. followed by 3 buffer washes.

F. Antigen Retrieval AR (should use the steamer because the microwave method is patented)

1. fill green tub with AR buffer of choice
2. fill slide holder with "dummy" slides and intersperse the test slides among the dummy slides so that the slide holder is full of slides-this allows even heating
3. cover and place in larger container with water that goes halfway up the tub, and heat for 5 minutes on high.
4. check levels and repeat a second time, for 5 more minutes
5. remove slide container from microwave, (Careful-IT WILL BE HOT) remove lid and let the container sit on counter top for 20 minutes, to cool
6. TBST and BMRUAD x3

G. IMMUNOSTAINING:

1. Overlay sections with CSA kit blocking solution to block non-specific protein binding
2. Overlay sections with Reagents 1 (Primary antibody used at predetermined dilution) or negative reagent controls) diluted in blocking reagent of choice, and incubate in humidified chamber for the specified length of time. Overnight incubation allows better penetration of antibody into paraffin sections. Remember to coverslip over the reagent on the section, so that the sections will stay covered with reagent, all throughout the incubation and will not dry out at any time, because if any dryign occurs, this will contribute to high background.
3. Repeat washes, with washes in between each step. These will involve, overlaying biotinylated secondary, followed by previously made ABC complex, follwed by Amplification reagent (Phenolic biotinylating enzyme), followed by labeled Streptavidin, followed by substrate, followed by nuclear counterstain, follwed by coverslipping.

Follow Kit instructions

N.B. For the amplification to work well without a high background the wash buffer has to have an increased concentration of salt, as referred to in the CSA (DAKO Cytomaton) kit instructions.

1. BSA-Sigma Cat. No. A2153
2. CSA Kit-DAKO Cat. No. K1500
3. or if you prefer fluorescence, you may plan to use either the TSA kit from NEN, or use a fluorescent labeled streptavidin in the last step after the amplification
4. green staining dish (VWR Cat.# 25608-904)
5. white staining dish VWR Cat.# 25608-906
6. slide holder (VWR Cat# 25608-868)
7. PBS buffer phosphate buffered saline pH 7.4
8. TBS buffer :

   TBST= 50mM Tris/ 150mM NaCl/0.05% Tween 20

   TBST for CSA 50mM Tris, 300 mM NaCl/ 0.05%Tween 20

   a. TBS powder concentrate: Biogenex Cat. # HK098-5K

   b. TBS powder concentrate: DAKO Cat.# S1968

9. Tween 20 (Polyoxyethylene-Sorbitan Monolaureate)-Sigma Cat. No. P1379
10. Commercially available Antigen retrieval buffer from Biogenex or from Dako
11. OR Prepare 0.01 M Citrate Buffer, pH 6.0 (41 mL 0.1 M Sodium Citrate, 9 mL 0.1 M Citric Acid + 450 mL MQ Water) in 350 W Microwave - 5 min, twice see procedure outlined above
12. Avidin/Biotin Blocking Kits:

    a. Vector Cat. No. SP-2001

    b.Dako Cat. No. X0590

    c.Sigma Avidin Cat no A 9275

    d. Sigma Biotin Cat no B4501

13. Mouse Anti Human Vimentin-DAKO Cat. No. N1521 prediluted
14. Mouse IgG monoclonal supernatant from P3X63 Ag8, usually at 5 ug/ml, negative reagent control
15. Rabbit anti human vWf Dako Cat # N1505
16. Rabbit IgG Dako Cat.# X0936 negative rabbit reagent control, use at 5 ug/ml
17. AEC substrate:

    a. Vector Cat. No. SK-4200

    b.Biogenex Cat # HK092-5K

18. 30% H2O2-Fisher Cat. No. H325-100
19. Aquamount-Fisher Cat. No. BM-01
20. Mayer's Hematoxylin- Sigma Cat. No. MH532-1L