Dear Alexandra, Please look at here

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2651088/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC373207

http://www.ihcworld.com/_protocols/antibody_protocols/cd31_pharmingen.htm

http://www.abcam.com/ps/pdf/protocols/double%20immunofluorescence%20-simultaneous%20protocol.pdf

http://jhc.sagepub.com/content/54/4/385.full.pdf+html

look at this protocol;

CD31 (PECAM-1) Immunofluorescence Staining Protocol for Paraffin Sections

Solutions and Reagents

A. 10X Phosphate Buffered Saline (PBS):

To prepare 1 liter,

Na2HPO4 ------------------ 10.9 g

NaH2PO4 ------------------ 3.2 g

NaCl ------------------------ 90 g

Distilled water ----------- 1000 ml

Mix to dissolve and adjust pH to 7.4

B. Antigen Retrieval Solution:

Pronase Stock Solution (1% pronase in PBS):

Pronase ------------------------- 100 mg

PBS ------------------------------- 10 ml

Mix to dissolve. Aliquot and store at – 20 C

Pronase Working Solution (0.1% in PBS):

Pronase Stock Solution (1%) --------- 1 ml

PBS ----------------------------------------- 10 ml

C. Blocking Solution:

2% Normal Rabbit Serum in PBS:

To prepare 100 ml

Normal rabbit serum ------- 2 ml

PBS ---------------------------- 100 ml

Mix to dissolve.

D. Primary Antibody:

Rat anti-mouse CD31 (PECAM-1) (PharMingen Cat# 553370). Optimal dilution 1:100 in PBS.

E. Secondary Antibody:

Rabbit anti-rat IgG (H+L), biotinylated (Vector Laboratories, Cat# BA-4000). Optimal dilution 1:400.

F. FITC-Streptavidin Reagent:

Vector Laboratories, Cat# SA-5001. Optimal dilution 1:50 in PBS.

G. PI Stock Solution (1mg/ml or 1.5 mM):

To prepare, add 1 mg PI (Propidium Iodide) to 1 ml distilled water. Store stock solution at 4 C (or aliquot and store at –20 C), protected from light. When handled properly, solutions are stable for at least six month.

H. RNase A Stock Solution (1mg/ml):

To prepare, add 1 mg RNase A to 1 ml distilled water. Aliquot and store at –20 C freezer.

I. PI Working Solution (1 ug/ml PI and 10 ug/ml RNase A in PBS):

To prepare, add 2 ul PI Stock Solution and 20 ul RNase A Stock Solution to 2 ml PBS.

Procedure

1. Deparaffinize sections in xylene for 3x5min.

2. Hydrate with 100% ethanol for 2x5min.

3. Hydrate with 95% ethanol for 2x5min.

4. Rinse in distilled water.

5. Antigen Retrieval: Use pronase or trypsin pretreatment antigen retrieval method.

6. Rinse sections in PBS for 1x5min.

7. Blocking: incubate sections with 2% normal rabbit serum in PBS for 20 minutes

8. Rinse in PBS for 1x2min.

9. Primary antibody: incubate sections with rat anti-mouse CD31 diluted 1:100 in PBS for 1 hour at room temperature or overnight at 4 C.

10. Rinse in PBS 3x5 min.

11. Secondary antibody: incubate sections with biotinylated rabbit anti-rat IgG diluted 1:400 in PBS for 30 minutes at room temperature.

12. Rinse in PBS for 3x5min.

13. Incubate sections in FITC-streptavidin for 30 minutes, protecting the slide from light.

14. Rinse 3x5min in PBS.

15. Counterstain with PI Working Solution for 20 minutes at 37 C.

16. Rinse 3x5min in PBS.

17. Coverslip with aqueous mounting medium and seal with nail polish.

18. Observation using a fluorescence microscope with appropriate filters. Store slides in the dark at 4 °C.

Results

1. Staining pattern -------------------------------endothelial cells

2. CD31 positive sites --------------------------- green

3. Nuclei ------------------------------------------ red

All the best

Ali

You have to give more information about the species you work with and the protocol you have tried until now. Do you want to stain immunofluorescence or chromogen? etc.

https://www.emsdiasum.com/microscopy/technical/datasheet/14654a.aspx

The staining protocol mentioned by Ali does not work as I don't have paraffin embedded tissue but GMA. 

I work with mouse tissue and I tried the following antibodies:

CD31 (sc-1506 Santa Cruz), CD31 (MON1149 Monosan), CD31 (550274 BD), CD34 (Ab8158 Abcam), CD34 (09431D PharMingen), GLS-1 (Vectorlabs), CD31-biotin (13-0311-85 Ebiosciences). 

I tested two different trypsin concentrations and incubation times and always tested the antibody 1:50:

Preparations:

-       Dry slides at 37 °C.

-       Make the 0.01% trypsin solution (40 ul trypsin stock (0.25%) + 960 ul CaCl2/TRIS/HCL stock).

Make the 0.1% trypsin solution (400 ul trypsin stock + 600 ul CaCl2/TRIS/HCL stock)

-       Put the solutions at 37 °C.

-       Put a slide holder at 37 °C.

-       Put PBS at 37 °C.

-       Put primary antibody solution at 37 °C.

-       20 ul Antibody + 980 ul PBS (+ 1% BSA)

Method:

-       Incubate 5 or 10 min in 0.01% trypsin (in 0.1% CaCl2 in 0.1 M TRIS/HCL, pH 7.8) or 0.1% trypsin at 37 °C.

-       Wash 3 times with PBS

-       Incubate for 2 hours at 37 °C with CD31 Antibody eBioscience (13-0311-85, rat-anti-mouse) 1:50 (in PBS + 1% BSA) or negative control with just PBS.

-       Wash 3 times with PBS.

-       Incubate 30 minutes at 20 °C endogene peroxidase block (5 ml Methanol + 12.5 ul 30% H2O2).

-       Wash 3 times with PBS.

-       Incubate 5 minutes at 20 °C with goat serum (10%).

-       200 ul NGS + 1800 ul PBS

-       Incubate 45 minutes at 20 °C with Streptavidine-HrP (1:100) diluted in PBS + 1% BSA.

-       20 ul antibody + 1980 ul PBS (+1% BSA)

-       Wash 3 times with PBS

-       Incubate 5-10 minutes with DAB

-       40 ul DAB + 1.6 ml PBS + 1.6 ul 30% H2O2

-       Add demi water to stop the reaction.

-       Incubate really short (20 sec) in Haematoxyline

-       Cover with Aquatex

I hope it is more clear now, don't have a preference for immunofluorescence or chromogen, as long as it works. 

In addition, we fixated the tissue with 2% PFA for 3 hours than changed to 6% sucrose overnight. Aceton for 1 hour with several changes. Overnight in technovit 8100, next day adding the hardener and embedding in mold. All steps are conducted at 4C. 

You work with an biotinylated antibody as primary. Therefore you have no amplification through a secondary antibody. This might result in a too low sensitivity for the used dilution

The described antibody is made for flowcytometric not for IHC as the vendor states. It might be unsuccessful for this application.

Are the other primaries also biotinylated? If not, a biotinylated secondary is necessary to get any result.

No only that one was biotinylated. Some of the others were also described in literature for resin staining. 

Have you used the same protocol as you described above for all tested antibodies?

What is the function of the "goat-serum", if you don't use any secondary antibody? Usually the goat-serum or normal-serum is used to block unspecific binding of the secondary antibody. The normal-serum is from the same species of the secondary (eg. goat-anti-rat).

Fixation: For me 3 hours in 2% PFA sounds rather short. It is hard to say, if the antigen is sufficiently preserved or if it is influenced by the following reagens (aceton-fixation). - and if this effect inhibits binding of the antibody or not. 

Did this protocol work for other researchers in your group, or others previously stained antigens?

I used kind of the same protocol for all the antibodies, only the secondary antibody was different. (sorry in the case of this antibody the goat serum did not had any function, but for the other antibodies it was correlated to the species of the secondary). 

We have a insulin staining working with this exact same protocol.