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Immunofluorescence
Immunofluorescence: Common staining protocol
Paraffin sections
1- Deparaffinization / rehydration
- 30 min 50°C
- 3 x 3 min Xylol RT
- 3 min EtOH 96%
- 3 min EtOH 96%
- 3 min EtOH 80%
- 3 min EtOH 70%
- 30 min with PBS
2- Antigen Retrieval
- Autoclave: 10 min ,121°C
Buffer: Tris-HCL 10 mM, Tween 0,05%, pH9
(or alternatively use a microwave and heat it for 6 minutes without
cooking in order to avoid concentration changes)
- Let cool down in the buffer for 30 min
- 10 min PBS/Twin
- 10 min Glycin buffer (200ml dH2O + 4g Glycin)
- 10 min PBS/Twin
Immunofluorescence Staining
1- Encircle the tissue with Dako Pen
2- 30 min in PBS/Twin + 5% Goat serum (or serum from the same species as II ab)
(blocking)
Indirect Staining
3- 1h primary antibodies at RT (use shaking with rotation platform)
4- 2 x3 min with PBS/Twin
5- 30 min fluorochrom conjugated secondary antibodies at RT (use shaking).
From now on, please protect the slides from light!!
6- 2 x 3 min with PBS/Twin
Direct Staining
5- 1h direct conjugated antibodies at RT in PBS/Twin (shake)
6- 2 x 3 min with PBS/Twin
7- Counterstaining by use DAPI (1:100 000) in stabilize from DAKO, 5 min
8- 2 x 3 min with PBS/Twin
9- Cover slip slides by using a mounting medium that supports the endurance of
fluorescence (e.g. GelTol Aqueous Mounting Medium – Thermo/Shannon)
10- 20 min at RT (to harden the mounting medium)
Paraffin sections
1- Deparaffinization / rehydration
- 30 min 50°C
- 3 x 3 min Xylol RT
- 3 min EtOH 96%
- 3 min EtOH 96%
- 3 min EtOH 80%
- 3 min EtOH 70%
- 30 min with PBS
2- Antigen Retrieval
- Autoclave: 10 min ,121°C
Buffer: Tris-HCL 10 mM, Tween 0,05%, pH9
(or alternatively use a microwave and heat it for 6 minutes without
cooking in order to avoid concentration changes)
- Let cool down in the buffer for 30 min
- 10 min PBS/Twin
- 10 min Glycin buffer (200ml dH2O + 4g Glycin)
- 10 min PBS/Twin
Immunofluorescence Staining
1- Encircle the tissue with Dako Pen
2- 30 min in PBS/Twin + 5% Goat serum (or serum from the same species as II ab)
(blocking)
Indirect Staining
3- 1h primary antibodies at RT (use shaking with rotation platform)
4- 2 x3 min with PBS/Twin
5- 30 min fluorochrom conjugated secondary antibodies at RT (use shaking).
From now on, please protect the slides from light!!
6- 2 x 3 min with PBS/Twin
Direct Staining
5- 1h direct conjugated antibodies at RT in PBS/Twin (shake)
6- 2 x 3 min with PBS/Twin
7- Counterstaining by use DAPI (1:100 000) in stabilize from DAKO, 5 min
8- 2 x 3 min with PBS/Twin
9- Cover slip slides by using a mounting medium that supports the endurance of
fluorescence (e.g. GelTol Aqueous Mounting Medium – Thermo/Shannon)
10- 20 min at RT (to harden the mounting medium)
11- Store at 4°C in darkness
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