|10X Wash Buffer, Phosphate Buffered Saline (PBS) 12528||35 ml|
|16% Formaldehyde, Methanol-Free 12606||10 ml|
|Immunofluorescence Blocking Buffer 12411||10 ml|
|Immunofluorescence Antibody Dilution Buffer 12378||20 ml|
All components in this kit are stable for at least 12 months when stored at the recommended temperature and left unused. Formaldehyde fixative should be used within one month once opened.
Upon receipt, #12528 and #12606 should be stored at room temperature. #12411 and #12378 should be stored at 4°C.
NOTE: Please refer to the antibody data sheet to determine if it is validated and approved for use on cultured cell lines (IF-IC) or frozen tissue sections (IF-F) and for information regarding appropriate antibody dilution.
Some primary antibodies may require methanol fixation or permeabilization, which will be noted on the data sheet. Methanol is not included in this kit.
16% Formaldehyde, Methanol-Free: (#12606) Use fresh, dilute to 4% with 1X Wash Buffer. Leftover diluted material should be discarded.
NOTE: The screw cap allows for the entire vial contents to be used at once. To extend the product’s shelf-life, small volumes should be extracted by piercing the silicone top with a needle and syringe. Store protected from light and use within one month after opening.
Fluorochrome-conjugated secondary antibody (if applicable).
NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution provides the strongest specific signal with the least background for your sample.
Cultured Cell Lines (IF-IC)
NOTE: Cells should be cultured, treated, fixed, and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid and cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X Wash Buffer.
NOTE: Formaldehyde is toxic, use only in fume hood.
IMPORTANT: If noted on the primary antibody data sheet, use ice-cold 100% methanol in lieu of formaldehyde as the fixative.
Frozen/Cryostat Sections (IF-F)
NOTE: Unless otherwise noted, all subsequent incubations should be carried out at room temperature in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading. Do not allow slides to dry at any time during this process.
Methanol Permeabilization Step (if applicable): Aspirate wash buffer and cover specimen to a depth of 3-5 mm with ice-cold 100% methanol. Incubate for 10 min at -20°C, and then rinse in 1X Wash Buffer for 5 min.
NOTE: This step is only required if noted on the primary antibody data sheet.
Rinse three times with 1X Wash Buffer for 5 min each.
NOTE: If using a fluorochrome-conjugated primary antibody, such as an Alexa Fluor® fluorochrome antibody, then skip to (Section C, Step 9).
posted December 2017
Protocol Id: 1664
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