Immunofluorescence and immunohistochemistry

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
• 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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