3. MATERIALS AND METHODS
3.3. Immunhistology
3.3.2. Immunhistological stains
Antibody: Mouse anti-cytokeratin pan (clone: Lu5)
Provenance: Neomarkers Dunn Labortechnik GmbH, Asbach Sections: Deparaffinized sections (see 3.3.1.1.)
Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Pre-processing with Protease (see 3.3.1.3.1.) 4) Insert slides in Shandon Racks
5) Blocking serum for 10 min at room temperature, horse serum.
6) Incubate primary antibody over night at 4°C in the refrigerator
MATERIALS AND METHODS 47 Dilute the antibody in TBS with1 % BSA (see 3.3.1.4.)
7) Rinse 3 x 5 min in TBS buffer
8) Incubate the secondary antibody for 30 min at room temperature, Biotinylated Horse anti-Mouse IgG (H+L)
Preparation: 9 µl for 1 ml TBS with 1 % BSA buffer 9) Rinse the sections 3 x 5 min in TBS buffer
10) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl Avidin + 9 µl Biotin for 1 ml TBS buffer Attention! It has to be prepared 30 min before use
11) Then as above (see 3.3.1.5.)
3.3.2.2. Vimentin
Antibody: Mouse anti-vimentin (clone V9)
Provenance: Dako Cytomalion GmbH, Hamburg Sections: Deparaffinized sections (see 3.3.1.1.)
Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Insert slides in Shandon Racks
4) Blocking serum for 10 min at room temperature, horse serum.
5) Incubate primary antibody over night at 4°C in the refrigerator Dilute the antibody in TBS with1 % BSA (see 3.3.1.4.)
6) Rinse 3 x 5 min in TBS buffer
7) Incubate the secondary antibody for 30 min at room temperature, Biotinylated Horse anti-Mouse IgG (H+L)
Preparation: 9 µl for 1 ml TBS with1 % BSA buffer 8) Rinse the sections 3 x 5 min in TBS buffer
9) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl Avidin + 9 µl Biotin for 1 ml TBS buffer Attention! It has to be prepared 30 min before use
10) Then as above (see 3.3.1.5.)
MATERIALS AND METHODS 48 3.3.2.3. Actin
Antibody: Mouse anti-actin (clone HHF-35)
Provenance: Dako Cytomation GmbH, Hamburg Sections: Deparaffinized sections (see 3.3.1.1.)
Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Insert slides in Shandon Racks
4) Blocking serum for 10 min at room temperature, horse serum.
5) Incubate primary antibody over night at 4°C in the refrigerator Dilute the antibody in TBS with 1 % BSA (see 3.3.1.4.)
6) Rinse 3 x 5 min in TBS buffer
7) Incubate the secondary antibody for 30 min at room temperature, Biotinylated Horse anti-Mouse IgG (H+L)
Preparation: 9 µl for 1 ml TBS with 1 % BSA buffer 8) Rinse the sections 3 x 5 min in TBS buffer
9) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl Avidin + 9 µl Biotin for 1 ml TBS buffer Attention! It has to be prepared 30 min before use
10) Then as above (see 3.3.1.5.)
3.3.2.4. Desmin
Antibody: Mouse anti-desmin (clone D33)
Provenance: Dako Cytomation GmbH, Hamburg Sections: Deparaffinized sections (see 3.3.1.1.)
Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Insert slides in Shandon Racks
4) Blocking serum for 10 min at room temperature, dilute horse serum 1:5 in TBS 20 min 5) Incubate primary antibody over night at 4°C in the refrigerator
Dilute the antibody in TBS (see 3.3.1.4.) 6) Rinse 3 x 5 min in TBS buffer
MATERIALS AND METHODS 49 7) Incubate the secondary antibody for 30 min at room temperature, Biotinylated Horse anti-Mouse IgG (H+L)
Preparation: 9 µl for 1 ml TBS buffer 8) Rinse the sections 3 x 5 min in TBS buffer
9) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl Avidin + 9 µl Biotin for 1 ml TBS buffer Attention! It has to be prepared 30 min before use
10) Then as above (see 3.3.1.5.)
3.3.2.5. Neurofilament
Antibody: Mouse anti-neurofilament (clone 2F11) Provenance: Dako Cytomation GmbH, Hamburg Sections: Deparaffinized sections (see 3.3.1.1.)
Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Insert slides in Shandon Racks
4) Blocking serum for 10 min at room temperature, horse serum.
Incubate primary antibody over night at 4°C in the refrigerator.
Dilute the antibody in TBS with 1 % BSA (see 3.3.1.4.) 5) Rinse 3 x 5 min in TBS buffer
6) Incubate the secondary antibody for 30 min at room temperature, Biotinylated Horse anti-Mouse IgG (H+L)
Preparation: 9 µl for 1 ml TBS buffer 7) Rinse the sections 3 x 5 min in TBS buffer
8) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl Avidin + 9 µl Biotin for 1 ml TBS buffer Attention! It has to be prepared 30 min before use
9) Then as above (see 3.3.1.5.)
3.3.2.6. Glial fibrillary acidic protein (GFAP) Antibody: Rabbit anti-GFAP (rabbit polyclonal)
Provenance: Dako Cytomation GmbH, Hamburg Sections: Deparaffinized sections (see 3.3.1.1.)
MATERIALS AND METHODS 50 Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Insert slides in Shandon Racks
4) Blocking serum for 10 min at room temperature, swine serum Incubate primary antibody over night at 4°C in the refrigerator Dilute the antibody in TBS with 20% SS (see 3.3.1.4.)
5) Rinse 3 x 5 min in TBS buffer
6) Incubate the secondary antibody for 30 min at room temperature, Goat anti-Rabbit IgG Preparation: 9 µl for 1 ml TBS with 20% SS
7) Rinse the sections 3 x 5 min in TBS buffer
8) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl A+ 9 µl B dilute 1 ml TBS
9) Then as above (see 3.3.1.5.)
3.3.2.7. Melan A
Antibody: Mouse anti-melan A (clone A103)
Provenance: Dako Cytomation GmbH, Hamburg Sections: Deparaffinized sections (see 3.3.1.1.)
Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Pre-processing with citrate (see 3.3.1.3.2.) 4) Insert slides in Shandon Racks
5) Blocking serum for 10 min at room temperature, horse serum Incubate primary antibody over night at 4°C in the refrigerator Dilute the antibody in TBS with 1 % BSA (see 3.3.1.4.) 6) Rinse 3 x 5 min in TBS buffer
7) Incubate the secondary antibody for 30 min at room temperature, Biotinylated Horse anti-Mouse IgG (H+L)
Preparation: 9 µl for 1 ml TBS buffer 8) Rinse the sections 3 x 5 min in TBS buffer
9) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl Avidin + 9 µl Biotin for 1 ml TBS buffer
MATERIALS AND METHODS 51 Attention! It has to be prepared 30 min before use
10) Then as above (see 3.3.1.5.)
3.3.2.8. Factor VIII
Antibody: Rabbit anti-factor VIII (rabbit polyclonal) Provenance: Dako Cytomation GmbH, Hamburg Sections: Deparaffinized sections (see 3.3.1.1.)
Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Pre-processing with Protease (see 3.3.1.3.1.) 4) Insert slides in Shandon Racks
5) Blocking serum for 10 min at room temperature, swine serum Incubate primary antibody over night at 4°C in the refrigerator.
Dilute the antibody in TBS with 20% SS (see 3.3.1.4.) 6) Rinse 3 x 5 min in TBS buffer
7) Incubate the secondary antibody for 30 min at room temperature, Biotinylated Goat anti-Rabbit IgG
Preparation: 9 µl for 1 ml TBS with 20% SS 8) Rinse the sections 3 x 5 min in TBS buffer
9) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl A + 9 µl B dilute 1 ml TBS
10) Then as above (see 3.3.1.5.)
3.3.2.9. MHC II
Antibody: Mouse anti-human HLA-DR, alpha chain (clone TAL.1B5) Provenance: Dako Cytomation GmbH, Hamburg
Sections: Deparaffinized sections (see 3.3.1.1.) Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Pre-processing with citrate (see 3.3.1.3.2.) 4) Insert slides in Shandon Racks
MATERIALS AND METHODS 52 5) Blocking serum for 10 min at room temperature, rat serum 10% in TBS
Incubate primary antibody over night at 4°C in the refrigerator Dilute the antibody in TBS (see 3.3.1.4.)
6) Rinse 3 x 5 min in TBS buffer
7) Incubate the secondary antibody for 30 min at room temperature, Rat anti-Mouse IgG Preparation: dilute 1:1000 in TBS
8) Rinse the sections 3 x 5 min in TBS buffer
9) Incubate the mouse PAP complex for 30 min at room temperature Preparation: dilute 1:500 in TBS
10) Then as above (see 3.3.1.5.)
3.3.2.10. Lysozyme
Antibody: Rabbit anti-lysozyme (rabbit polyclonal) Provenance: Dako Cytomation GmbH, Hamburg Sections: Deparaffinized sections (see 3.3.1.1.)
Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Pre-processing with Protease (see 3.3.1.3.1.) 4) Insert slides in Shandon Racks
5) Blocking serum not applied
Incubate primary antibody over night at 4°C in the refrigerator Dilute the antibody in TBS/5 % dog serum (see 3.3.1.4.) 6) Rinse 3 x 5 min in TBS buffer
7) Incubate the secondary antibody for 30 min at room temperature, swine anti-rabbit IgG Preparation: dilute 1:100 in TBS with 5 % dog serum
8) Rinse the sections 3 x 5 min in TBS buffer
9) Incubate the PAP from rabbit for 30 min at room temperature Preparation: dilute 1:100 in TBS with 5 % dog serum 10) Then as above (see 3.3.1.5.)
3.3.2.11. CD3
Antibody: Rabbit anti-CD3 (clone pAb)
Provenance: Dako Cytomation GmbH, Hamburg
MATERIALS AND METHODS 53 Sections: Deparaffinized sections (see 3.3.1.1.)
Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Pre-processing with Protease (see 3.3.1.3.1.) 4) Insert slides in Shandon Racks
5) Blocking serum for 15 min at room temperature, dilute swine serum 1:5 in TBS Incubate primary antibody over night at 4°C in the refrigerator
Dilute the antibody in TBS/20 % SS (see 3.3.1.4.) 6) Rinse 3 x 5 min in TBS buffer
7) Incubate the secondary antibody for 30 min at room temperature, Biotinylated Goat anti-Rabbit IgG (H+L)
Preparation: 9 µl for 1 ml TBS buffer / 20 % SS 8) Rinse the sections 3 x 5 min in TBS buffer
9) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl Avidin + 9 µl Biotin for 1 ml TBS buffer Attention! It has to be prepared 30 min before use
10) Then as above (see 3.3.1.5.)
3.3.2.12. CD45R
Antibody: CD45R (clone B220 (Ly 5))
Provenance: Linaris GmbH, Bettingen Stadt Wertheim Sections: Deparaffinized sections from cat (see 3.3.1.1.) Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.):
2) Rinse for 5 min in TBS
3) Pre-processing with citrate (see 3.3.1.3.2.) 4) Insert slides in Shandon Racks
5) Blocking serum for 10 min at room temperature, horse serum Incubate primary antibody over night at 4°C in the refrigerator Dilute the antibody in TBS with 1 % BSA (see 3.3.1.4.) 6) Rinse 3 x 5 min in TBS buffer
7) Incubate the secondary antibody for 30 min at room temperature, Biotinylated Rabbit anti-Rat IgG (H+L)
MATERIALS AND METHODS 54 Preparation: 9 µl for 1 ml TBS buffer
8) Rinse the sections 3 x 5 min in TBS buffer
9) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl Avidin + 9 µl Biotin for 1 ml TBS buffer Attention! It has to be prepared 30 min before use
10) Then as above (see 3.3.1.5.)
3.3.2.13. CD79α
Antibody: Mouse anti-B-cell CD79αcy (clone HM57) Provenance: Dako Cytomation GmbH, Hamburg Sections: Deparaffinized sections from cat (see 3.3.1.1.) Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Pre-processing with citrate (see 3.3.1.3.2.) 4) Insert slides in Shandon Racks
5) Blocking serum for 10 min at room temperature, horse serum Incubate primary antibody over night at 4°C in the refrigerator Dilute the antibody in TBS with 1 % BSA (see 3.3.1.4.) 6) Rinse 3 x 5 min in TBS buffer
7) Incubate the secondary antibody for 30 min at room temperature, Biotinylated Horse anti-Mouse IgG (H+L)
Preparation: 9 µl for 1 ml TBS buffer 8) Rinse the sections 3 x 5 min in TBS buffer
9) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl Avidin + 9 µl Biotin for 1 ml TBS buffer Attention! It has to be prepared 30 min before use
10) Then as above (see 3.3.1.5.)
3.3.2.14. Neuron Specific Enolase (NSE) Antibody: Anti-NSE (monoclonal antibody)
Provenance: “Zymed” Invitrogen GmbH, Karlsruhe Sections: Deparaffinized sections (see 3.3.1.1.)
MATERIALS AND METHODS 55 Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Pre-processing with citrate (see 3.3.1.3.2.) 4) Insert slides in Shandon Racks
5) Blocking serum for 10 min at room temperature, horse serum Incubate primary antibody over night at 4°C in the refrigerator Dilute the antibody in TBS with 1 % BSA (see 3.3.1.4.) 6) Rinse 3 x 5 min in TBS buffer
7) Incubate the secondary antibody for 30 min at room temperature, Biotinylated Horse anti-Mouse IgG (H+L)
Preparation: 9 µl for 1 ml TBS buffer 8) Rinse the sections 3 x 5 min in TBS buffer
9) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl Avidin + 9 µl Biotin for 1 ml TBS buffer Attention! It has to be prepared 30 min before use
10) Then as above (see 3.3.1.5.)
3.3.2.15. Synaptophysin
Antibody: Anti-synaptophysin (clone SY38)
Provenance: Dako Cytomation GmbH, Hamburg Sections: Deparaffinized sections (see 3.3.1.1.)
Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Pre-processing with citrate (see 3.3.1.3.2.) 4) Insert slides in Shandon Racks
5) Blocking serum for 10 min at room temperature, horse serum Incubate primary antibody over night at 4°C in the refrigerator Dilute the antibody in TBS with 1 % BSA (see 3.3.1.4.) 6) Rinse 3 x 5 min in TBS buffer
7) Incubate the secondary antibody for 30 min at room temperature, Biotinylated Horse anti-Mouse IgG (H+L)
Preparation: 9 µl for 1 ml TBS buffer
MATERIALS AND METHODS 56 8) Rinse the sections 3 x 5 min in TBS buffer
9) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl Avidin + 9 µl Biotin for 1 ml TBS buffer Attention! It has to be prepared 30 min before use
10) Then as above (see 3.3.1.5.)
3.3.2.16. S100
Antibody: Anti-S100 (rabbit polyclonal)
Provenance: Dako Cytomation GmbH, Hamburg Sections: Deparaffinized sections (see 3.3.1.1.)
Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Insert slides in Shandon Racks
4) Blocking serum for 30 min at room temperature, dilute swine serum 1:5 in TBS Incubate primary antibody over night at 4°C in the refrigerator
Dilute the antibody in TBS (see 3.3.1.4.) 5) Rinse 3 x 5 min in TBS buffer
6) Incubate the secondary antibody for 30 min at room temperature, Swine anti-Rabbit IgG Preparation: 9 µl for 1 ml TBS buffer
7) Rinse the sections 3 x 5 min in TBS buffer
8) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl Avidin + 9 µl Biotin for 1 ml TBS buffer Attention! It has to be prepared 30 min before use
9) Then as above (see 3.3.1.5.)
3.3.2.17. MAC387
Antibody: Anti-Myeloid/Histocyte Antigen (clone MAC387) Provenance: Dako Cytomation GmbH, Hamburg Sections: Deparaffinized sections (see 3.3.1.1.)
Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Pre-processing with Protease (see 3.3.1.3.1.)
MATERIALS AND METHODS 57 4) Insert slides in Shandon Racks
5) Blocking serum for 30 min at room temperature, rat serum 1:10 dilute in TBS Incubate primary antibody over night at 4°C in the refrigerator
Dilute the antibody in TBS (see 3.3.1.4.) 6) Rinse 3 x 5 min in TBS buffer
7) Incubate the secondary antibody for 30 min at room temperature, Swine anti-Rabbit IgG Preparation: 9 µl for 1 ml TBS buffer
8) Rinse the sections 3 x 5 min in TBS buffer
9) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl Avidin + 9 µl Biotin for 1 ml TBS buffer Attention! It has to be prepared 30 min before use
10) Then as above (see 3.3.1.5.)
3.3.2.18. Smooth muscle actin
Antibody: Mouse anti-smooth muscle actin (clone 1A4) Provenance: Dako Cytomation GmbH, Hamburg Sections: Deparaffinized sections (see 3.3.1.1.)
Procedure:
1) Inhibition of the endogeneous peroxidase (see 3.3.1.2.) 2) Rinse for 5 min in TBS
3) Pre-processing with citrate (see 3.3.1.3.2.) 4) Insert slides in Shandon Racks
5) Blocking serum for 10 min at room temperature, horse serum Incubate primary antibody over night at 4°C in the refrigerator Dilute the antibody in TBS with 1 % SS (see 3.3.1.4.)
6) Rinse 3 x 5 min in TBS buffer
7) Incubate the secondary antibody for 30 min at room temperature, Biotinylated Horse anti-Mouse IgG (H+L)
Preparation: 9 µl for 1 ml TBS buffer 8) Rinse the sections 3 x 5 min in TBS buffer
9) Incubate the ABC complex for 30 min at room temperature Preparation: 9 µl Avidin + 9 µl Biotin for 1 ml TBS buffer Attention! It has to be prepared 30 min before use
10) Then as above (see 3.3.1.5.)
RESULTS 58