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Immunofluorescence (Tryptase and PGP9.5) and immunohistochemistry (H4R)
Last updated date: Aug 24, 2022 Views: 547 Forks: 0
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Immunofluorescence on paraffin sections: Tryptase and PGP9.5
Protocol
- Bake slides 60C for 20min
- 3 changes of xylene, 5 min each (Removal of paraffin)
- 100% EtOH , 3 changes, 3 min each
- Rinse in 70 % EtOH, 3min
- Rinse in distilled water, 5min
- 0.1% Sudan Black B solution, 20min incubation RT
- Rinse with water a couple of times
- Antigen retrieval with Citric acid Buffer in the steamer 30 min
- Take out samples and cool down 30 min
- Rinse with TBS-T 3 times
- Draw a circle by PAP pen
- Block in the 5% BSA, 30 min RT
- Drain off excess of the blocking, do not wash
- Incubate with primary antibody, Mast cell tryptase (Abcam: ab151757, Rabbit), diluted 1:200, PGP9.5 (Abcam: ab10410, Guinea Pig), diluted 1:250 in 1% BSA, over night at 4C, approx.100-200 uL per slide
- Wash VERY WELL with TBS-T from squeeze bottle, 3x
- Incubate with secondary antibody, Donkey anti-rabbit (Invitrogen A31572, 555), diluted 1:1000, Donkey anti-guinea pig, (Jackson Immuno research 706-545-148, 488) diluted 1:500 in 1% BSA, 1h incubation RT
- Wash VERY WELL with TBS from squeeze bottle, 3x
- Wash VERY WELL with distilled water from squeeze bottle, 1x
- Coverslip with Dapi ProLong
Materials
Antibodies and Products
1st antibody: Mast cell tryptase (Abcam: ab151757, Rabbit), diluted 1:200
PGP9.5 (Abcam: ab10410, Guinea Pig), diluted 1:250
2nd antibody: Donkey anti-rabbit (Invitrogen A31572, 555), diluted 1:1000
Donkey anti-guinea pig, (Jackson Immuno research 706-545-148, 488) diluted 1:500
Dapi: ProLongTM Gold antifade regent with DPI (Invitrogen P36935)
10xTris Buffered Saline (TBS)
Trizma base (C4H11NO3) 24.2g
NaCl 80 g
Adjust pH to 7.6 with approx. 50-60 ml conc. HCl
Adjust final volume to 1000 ml with distilled water
1xTBS-T
10xTBS 100mL
distilled water 900mL
Add 1mL Tween 20, mix well
0.1% Sudan black B
Sudan Black B (sigma, 199664) 0.1g
70%EtOH 100mL
Keep in 4C. you can use this solution repeatedly
Citric acid Buffer
A: 0.1M citric acid
Citric acid (BDH, ACS231) 2.1g
dH2O 100mL
B: 0.1M sodium citrate (sigma, C3434) 14.7g
dH2O 500mL
Both A and B Keep in 4C.
Before antigen retrieval,
A: 4.5mL
B: 20.5mL
Adjust pH6.0 with 3M NaOH
Then dilute A+B solution with x10 of water (225mL)
5% BSA
BSA (sigma, B4287) 2.5g
PBS 50mL
Keep in 4C.
1% BSA
BSA (sigma, B4287) 0.5g
PBS 50mL
Keep in 4C.
Immunohistochemistry on paraffin sections: H4R
Protocol
- Bake slide 60C for 20min
- 3 changes of xylene, 5 min each (Removal of paraffin)
- 100% EtOH , 3 changes, 3 min each
- Rinse in 70 % EtOH, 3min
- Rinse in distilled water, 5min
- Antigen retrieval with Citric acid Buffer in the steamer 30 min
- Take out samples and cool down 30 min
- Rinse in distilled water for 5 minutes.
- 3 % Hydrogen peroxide in water, 5 min incubation RT
- Rinse in distilled water for 2 minutes.
- Protein blocking step. Incubate sections for 20 minutes with ready-to-use (2.5%) normal horse serum (NHS).
- Drain off excess of the blocking serum, do not wash
- Incubate with primary antibody – Histamine 4 Receptor: 20ug/ml diluted 1:100 in the 2.5% Normal Horse Serum, 1h RT.
- Wash with TBS-T 2x2min
- Incubate with ImmPRESS anti-Rabbit IgG (ready to use), 30 min
- Wash with TBS-T 2x2min
- Wash VERY WELL with distilled water from squeeze bottle, 1x
- Prepare ImmPACT DAB (1 drop ImmPACT DAB to 1mL ImmPACT DAB Diluent).
- Incubate at RT for 3-5 minutes
- Rinse with water, leave under the running tap water 5-10 min
- Stain with Gill’s hematoxylin, 20-30 sec.
- Rinse with water, leave under the running tap water 5-10 min
- Dehydrate 70 % EtOH, 100% EtOH 2x, xylene 2x
- Coverslip with Permount
Materials
Antibodies and Chemicals
1st antibody: Histamine H4R (Origine TA340671)
2nd antibody: ImmPRESS Anti-Rabbit HRP (VECTOR: MP-6401)
DAB: ImmPACT DAB (VECTOR, SK-4105)
VectaMount PermNENT Mounting Medium (VECTOR: H-5000-60)
10xTris Buffered Saline (TBS)
Trizma base (C4H11NO3) 24.2g
NaCl 80 g
Adjust pH to 7.6 with approx. 50-60 ml conc. HCl
Adjust final volume to 1000 ml with distilled water
1xTBS-T
10xTBS 100mL
distilled water 900mL
Add 1mL Tween 20, mix well
Citric acid Buffer
A: 0.1M citric acid
Citric acid (BDH, ACS231) 2.1g
dH2O 100mL
B: 0.1M sodium citrate (sigma, C3434) 14.7g
dH2O 500mL
Both A and B Keep in 4C.
Before antigen retrieval,
A: 4.5mL
B: 20.5mL
Adjust pH6.0 with 3M NaOH
Then dilute A+B solution with x10 of water (225mL)
- De Palma, G, Shimbori, C and Bercik, P(2022). Immunofluorescence (Tryptase and PGP9.5) and immunohistochemistry (H4R). Bio-protocol Preprint. bio-protocol.org/prep1875.
- De Palma, G., Shimbori, C., Reed, D. E., Yu, Y., Rabbia, V., Lu, J., Jimenez-Vargas, N., Sessenwein, J., Lopez-Lopez, C., Pigrau, M., Jaramillo-Polanco, J., Zhang, Y., Baerg, L., Manzar, A., Pujo, J., Bai, X., Pinto-Sanchez, M. I., Caminero, A., Madsen, K., Surette, M. G., Beyak, M., Lomax, A. E., Verdu, E. F., Collins, S. M., Vanner, S. J. and Bercik, P.(2022). Histamine production by the gut microbiota induces visceral hyperalgesia through histamine 4 receptor signaling in mice. Science Translational Medicine 14(655). DOI: 10.1126/scitranslmed.abj1895
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