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Immunofluorescence and immunohistochemistry
Last updated date: Oct 27, 2020 Views: 814 Forks: 0
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Protocol Immunofluorescence (IF)
- Cut tumor (freshly frozen tumors in OCT; NEG-50 #6502, ThermoScientific) sections of 8-10µM thickness using a Microtome
- Fix tumor sections into Microscope Slides (e.g., J1800AMNZ, ThermoFisher Scientific)
- Store slides at -80 °C
- Day before I.F., store Acetone at -20°C
- Permeabilize slides with 10’ of Acetone (cold)
- Wash slides 5’ with PBS using an easydip slide staining system (Milian) to remove OCT around tumor
- Place slides horizontally in a slide holder
- Add hydrophobic layer with Pap Pen (Dako, S2002) around the tumor (be careful on OCT surrounding the tissue. Pap pen may be washed away. In that case, re-add Hydrophobic layer around all the tissue)
- Add humidified towel under the slides
- Block with 20% FBS 30’RT
- 1x PBS 5’
- 1ary Antibodies
- 30’ Room.Temp. (or overnight at 4 °C)
- Spin down and filter the diluted solutions before using them
- Rat anti NKp46 1:100 and Goat anti CD31 1:200 (#AF3628, R&D system) 1:100 dilution in 2% BSA PBS
- Rabbit anti Foxp3 1:100 dilution in 2% BSA PBS
- Rabbit anti CD4 1:100 dilution in 2% BSA PBS
- Rabbit anti CD8 1:100 1:100 dilution in 2% BSA PBS
- Rabbit anti casp3 1:200 1:100 dilution in 2% BSA PBS
- Add Goat anti CD31 1:200 dilution in 2% BSA PBS in all above mentioned Antibodies
- 1ary Antibodies
- 2x PBS 5’
- 2ndary Antibodies:
- 30’ Room.Temp.
- Spin down and filter the diluted solutions before using them
- Donkey anti Goat Alexa 594 (A11058; Invitrogen Red) 1:500 + Donkey anti Rabbit Alexa 488 (A21206; Invitrogen, green) 1:200-500 (filter)
- For NK cell staining Donkey anti Rat 594 1:500 and Donkey anti-Goat 488 1:500 (Color Channel will be swapped in Image J)
- 2x PBS 5’
- DAPI 1:400 5’ RT in mQ or PBS
- 2x PBS 5’
- Vectashield Fluorescent Mounting Medium (S3023; Dako) 1 drop on each tumor + add Microscope cover glasses (ECN631-1574, VWR) _ get rid of air bubble with the help of a pipette tip
- Dry slides (usually at least 1h at Room Temperature)
- Read slides with Microscope with appropriate lasers (488nm, 594nm, DAPI)
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
- Puca, E(2020). Immunofluorescence and immunohistochemistry. Bio-protocol Preprint. bio-protocol.org/prep574.
- Weiss, T., Puca, E., Silginer, M., Hemmerle, T., Pazahr, S., Bink, A., Weller, M., Neri, D. and Roth, P.(2020). Immunocytokines are a promising immunotherapeutic approach against glioblastoma . Science Translational Medicine 12(564). DOI: 10.1126/scitranslmed.abb2311
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