- Home
- Protocols
-
Immunofluorescence (Tryptase and PGP9.5) and immunohistochemistry (H4R)
Last updated date: Aug 24, 2022 Views: 540 Forks: 0
Download PDF
Ask a
question
How to
cite
Favorite
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
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Post a Question
