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EF5 Procedure Manual

Note: Instructions on how to make all reagents needed below are available on the website under Recipes and Reagents or are commercially available.

  1. Preserving Tissue
    • Divide fresh tissue into desired workable sizes (we use nickel size or smaller)
    • Label and moisten small pieces of filter paper
    • Dab with OCT
    • Place tissue in OCT and move filter paper on to metal freezing disk on dry ice
    • As the tissue is freezing drizzle a tiny bit of OCT over the top
    • Place frozen samples in vial with a bit of ice/water to keep from drying out and store in -80°C freezer

  2. Obtaining Sections
    • Section at -25°C in a cryostat (cut away filter paper to mount in cryostat)
    • Place 10µ sections on poly-lysine coated slides
    • Obtain at least 5 good (minimal folds and rips), preferably sequential sections for simple staining procedure
    • Number slides to keep track of relative distances and depth in tissue (include "lost" sections in numbering system)
    • Note the time each section was taken
    • It is advisable to take at least two different "levels" for each tissue by cutting down 500µ between sampling.
    • When finished remount the tissue on a new piece of labeled tissue paper and note how many times it has been sectioned (S1, S2, S3..)

  3. Fixation (either with 4% Paraformaldehyde or Acetone)
  4. Note: All rinses are done in slide jars. Between all rinses and changes of solutions slides are to be blotted on a paper towel to eliminate carry-over between solutions.

    1. 4% Paraformaldeyde (PF) Fixation
      • Immediately after placing section on slide drop slide into slide jar containing 4%PF
      • Leave slides in 4%PF for 60±5 min.
      • 2X10 min. rinses in 1XPBS w/azides and thimerisol (these rinses are a minimum of 10 min, can be longer to allow slides to "catch up" to each other).
      • Blot away moisture around section and block (see below)
    2. Acetone Fixation
      • Immediately after placing section on slide, place slide on slide rack.
      • Leave slide to dry for at least 30 min.
      • 5 min. in ice cold acetone
      • Leave dry on slide rack for 30 min.

      Note: at this point you can store slide in a refrigerated slide box with dessicant or continue by blocking and staining.


  5. Blocking
    • Blot away moisture around section.
    • Circle section with PAP pen (hydrophobic pen).
    • Add block (for general EF5 staining use mouse block).
    • Place slides (4 at a time) in slightly wet 96 well plate-lids to keep moist (these are stackable. Use one empty one to serve as lid to the stack.)
    • Refrigerate overnight

  6. Staining Procedures
  7. Note: All rinses are done in slide jars. Between all rinses and changes of solutions slides are to be blotted on a paper towel to eliminate carry-over between solutions.

    ****All staining procedures and rinses are done in subdued light using cold reagents. Unless otherwise noted slides are refrigerated or kept on ice between steps.*****

    1. General EF5 Staining
    2. 5 slides necessary
      2 for EF5 regular stains (RS) with Cy3 conjugated ELK-351 antibody
      2 for EF5 competed stains (CS) with Cy3 conjugated ELK-351 antibody +
      EF5 durg
      1 for no stain (NS) which is left in block

      Note: see FAQ for explanation of the two control stains (CS and NS)

      For RS and CS only:
      • Remove block by tipping slide.
      • Dip in ttPBS
      • Blot away moisture
      • Add appropriate stain and let sit for 4-6 hours in trays covered in tin foil and refrigerated

      General EF5 Staining Rinses
      The parallel steps are to be completed simultaneously (All rinses should be in opaque or covered vials as slides are now light sensitive)

      Regular EF5 Stain (RS) Competed EF5 Stain No Stain
      Tip to remove stainTip to remove stainLeave in block
      Dip in ttPBSDip in ttPBSLleave in block
      45 min ttPBS rinse. ttPBS with EF5 pipetted onto horizontal slides. Let sit for 2 min then tip and for a total of 5 times. Leave the 5th rinse on the slide and return to tin foil covered tray for remainder of the 45 min rinse. Leave in block.
      45 min ttPBS rinse. Repeat above step for this rinse. Leave in block.
      45 min 1XPBS rinse. 45 min 1XPBS rinse. 45 min 1XPBS rinse.
      For Storage place in opaque vial containing 1%PF for a maximum of 2 weeks. For Storage place in opaque vial containing 1%PF for a maximum of 2 weeks. For Storage place in opaque vial containing 1%PF for a maximum of 2 weeks.
    3. Co-staining with EF5
    4. NOTE: These Co-stained slides are considered qualitative rather then quantitative as the EF5 staining may be affected by the co-stain.

      1. Endogenous Hoechst (Administered by blood permeation through vessels before tissue is removed from animal and stains for vessels. The tissue is then light sensitive so exposure to light must be kept at a minimum for the remainder of the procedure).
        • Section and fix with acetone as usual.
        • Photograph immediately after fixation.
        • Be very careful of photo-bleaching the section by exposing it to too much light while photographing.
        • After photography follow the general EF5 staining procedure to co-stain for EF5.

      2. Apoptosis (programmed cell death)
        • Section, fix (PF only) and rinse as usual.
        • Place slides in 70% ethanol in fridge (4°C) overnight.
        • Follow Chemicon International Apoptag Florescein in situ Apoptosis Detector Kit (S7110).
        • To Co-stain for EF5 then do 4X2 min. washes in 1XPBS at room temp.
        • Then block for one hour with mouse block and follow procedure for general EF5 staining (Note: Cannot use Cy3 because it is not compatible with FITC so must use Cy5 conjugated ELK-351)

      3. CD-31/PECAM (blood vessels), KI-67 (Proliferation), and MN75 (CA9)
        • Section, fix and rinse as usual
        • Make blocking reagent appropriate for specific stain and tissue type and block for 30-60 min at room temp.
        • Dip in ttPBS, blot and add primary antibody at 1:100 dilution of antibody to carrier and refrigerate overnight
        • Tip off anitbody, dip in ttPBS
        • 2x15 min rinses in ttPBS at room temp.
        • 1x15 min rinse in 1XPBS at room temp.
        • Blot away moisture and add secondary antibody (Cy5 usually) at 1:100 dilution of antibody in carrier for 45-60 min at room temp. From this point on the slides are light sensitve.
        • 2x15 min rinses in ttPBS at room temp.
        • 1x15 min rinse in 1XPBS at room temp.
        • Re-fix in 4%PF for 20-30 min. at room temp.
        • 2X10 min rinse in ttPBS at room temp.
        • 1X10 min rinse in 1XPBS at room temp.
        • Block with mouse block for 1hour at room temp.
        • Stain with Cy3 conjugated ELK-351 overnight in the refrigerator.
        • Rinse as usual (2X45 min in ttPBS,1X45 min in 1XPBS, store in 1%PF).

      4. BrdUrd and Iurd (cell proliferation)
        • Bromodeoxyuridine (BrdUrd, 5-bromo-2'deoxyuridne, molecular weight 307) or Iodouridine (Iurd) are given in vivo at 0.1g/kg (300µM) or in vitro tissue is treated with 10µM concentration.
        • Place slides in cold (4°C) 70% ethanol.
        • Pour off ethanol and rinse in ttPBS 3x15 min.
        • Add 50µL pepsin (5.5mg crude SIGMA 7000 pepsin in 2.75ml 0.5N HCl/1XPBS dilution of 1:10 ) (0.2mg/ml in 0.5N HCL with 10% of 10xPBS) to each slide and incubate for 20 min. at 37°C (warm room).
        • Tip off pepsin and add 150µL of Borax neutralizing buffer. Do this for 3 exchanges.
        • Chill slides on ice in trays for 1-2 min.
        • 2x10 min rinses in ttPBS.
        • Add appropriate block and incubate for 30 min. at 4°C
        • Dip in ttPBS.
        • Add primary antibody (1:50 concentration) and leave for 2 hours at 4°C.
        • 2x10 min rinse in ttPBS.
        • 1x30 min. rinse in ttPBS.
        • Add secondary antibody (1:50- usually FITC) for 30 min at 4°C
        • 2x10 min rinse in ttPBS.
        • 1x30 min. rinse in ttPBS.
        • Check staining ????
        • Re-fix in 4%PF to stabilize
        • Counterstain nuclei with 20µM Hoechst 33342 (2.8mg bis benzamine/ mL salime or 1xPBS and dilute conc. 40µL in 20mL 1xPBS).
        • Block with mouse block for 1 hour and stain for EF5 overnight in the refrigerator.
        • Rinse and store as usual.

Comments? Questions? E-mail kochc@mail.med.upenn.edu