Immunochemistry for Computational Molecular Phenotyping

  1. Fixation

    • 2.5% (250mM) glutaraldehyde/1% (333 mM) formaldehyde
    • 0.1 M phosphate (PB) or cacodylate buffer (CB) pH 7.4
    • 3% sucrose
    • 1 mM MgSO4

    Fix 15 m (retina) or longer depending on tissue thickness. Store in fixative indefinitely.

  2. Dehydration and Infiltration

    • 0.1 M phosphate buffer, pH 7.4 (PB)
    • 100% Acetone
    • 100% anhydrous MeOH and 75%, 85%, 95% MeOH in dH2O
    • DMP30
    • Eponate resin
    • DDSA


      Mixing Active Resin

    • Mix resin fresh or thaw from freezer. Work quickly to prevent components from hydrating.
    • Wear gloves (contact dermatitis). Prepare in a hood.
    • Mix 2 volumes DDSA and 1 volume Eponate gently by hand with a spatula until the solution is clear (2-5 min)
    • Add 0.6 ml DMP30 per 30 ml resin; Mix gently as before until clear (2-5 min). Solution will darken as the activated epoxide begins to bind oxygen.
    • Use immediately or store in 30-60 cc syringe in the freezer. Expel all air before freezing and plug syringe with a wooden dowel. Do not cap with a needle or plastic cover as they leak air.


      Dehydrating samples < 1 mm thickness( vol 5 ml unless noted otherwise)

    • 10 m PB
    • 10 m each 75%, 85%, 95% MeOH in dH2O
    • 2 x 10 m 100% MeOH
    • 2 x 10 m 100% Acetone



    • 60 m 50% resin in acetone
    • 4-20 h 75% resin in acetone
    • 60 m 100% resin
    • Embed samples in fresh resin in molds and cure at 60-65 deg C overnight

  3. Sectioning
    • Section serially at 40-200 nm
    • Float sections on dH2O droplets
    • Place sections on 12-well Cel-Line HTC® array slides in an N-pattern.
    • Dry at 80°C & store indefinitely

  4. Probing
      Basic Working Reagents

    • PB = 0.1 M phosphate buffer, pH 7.4
    • PBT = PB + 0.05% thimerosal, pH 7.4
    • 1% GSPBT = 1% goat serum in PBT
    • 2° Ab = 1 nm Gold conjugated or fluorophore conjugated IgG
    • Mounting medium
    • Prepare a 100X dilution of a selected Signature Immunologics Series 100 Antiserum in 1% GSPBT or
    • Prepare a specified dilution of a selected lab antiserum in 1% GSPBT



    • Deplasticize array slides
        Stock Solutions

      • 100% Anhydrous Ethanol (EtOH)
      • 100% Anhydrous Methanol (MeOH
      • Na methoxide (Sigma-Aldrich) or aged Na ethoxide:
      • Aged Na ethoxide: Place ( 1 inch of NaOH pellets in a heavy glass 500-1000 ml bottle with a glass stopper covered in Parafilm. Cover the pellets with ( 3 inches of EtOH, seal and place in the hood. Shake daily until the solution becomes dark brown and syrupy (7-14 d), whence it is ready for use. Add replacement EtOH as the stock is used. Mark as an extremely hazardous, caustic reagent.
      • Na Methoxide: 50:50 Na methoxide and MeOH. Mark as an extremely hazardous, caustic reagent.



      • Working solution: Dilute stock Na methoxide/ethoxide 1:4 v/v in anhydrous EtOH.
      • Place slides into glass or steel carriers (not plastic) and immerse in the working solution. The etching time is 14 min/μm and is linear (e.g. 30 min for 2 μm, 7 min for 500 nm, 1 min for gold 90 nm sections). Over-etching is not a problem. We routinely etch 200 nm sections for 7 min. The working solution can be used for several days if kept anhydrous.
      • Drain the excess solution by blotting the carrier onto a stack of paper towels.
      • Immediately immerse in 100% EtOH or MeOH and wash 2 min. Repeat 3X with fresh alcohols.
      • Rinse slides 5 min in running cool tap water
      • Dip slides in dH2O and air dry.


    • Deosmicate if necessary: Treat osmicated sections on glass slides 10 min with fresh 1% Na metaperiodate in deionized water, rinse 1 min in 0.1 M phosphate buffer (pH 7.4), dip in deionized water & dry. Heavily osmicated tissues may need longer or a 2-step protocal. Perform under a hood and observe all safety precautions for using oxidizing agents.
    • Probe at room temperature with antiserum 4 hours to overnight at 25 μl/well. Cover with an inverted glass staining dish sealed with plastic wrap to prevent evaporation.
    • Flick off antiserum. Dip once in 0.1 M PB to rinse off excess and wash 10 minutes 0.1 M PB; use plastic mailing cassettes that hold 5 slides (about 15 ml of solution).
    • Dip slides in deionized water and dry with an air canister. Incubate 60 min with 25 microliters/well of 2° Ab in 1% GSPBT (e.g. a 1 nm gold goat anti-rabbit IgG or a fluorophore conjugated anti-rabbit IgG).
    • Flick off 2° Ab. Dip in 0.1 M PB to rinse off excess. Wash in PB 1 hour in plastic cassettes.
    • Dip in dH2O and dry with an air canister. Mount in a medium of your choice if using fluorescence. Otherwise, go to visualization.

  5. Visualization
      Stock Solutions & Equipment

    • Stock A = 114 mg citric acid + 342 mg sodium citrate in 6 ml deionized water (make fresh – do not store)
    • Stock B = 0.5 g hydroquinone in 15 ml deionized water (make fresh – do not store)
    • Stock C = 1% aqueous silver nitrate (may be stored indefinitely at room temperature wrapped in foil – keep dark)
    • Working solution = 5 ml A + 1 ml B + 1 ml C, in that order, made up immediately before use
    • Stop solution = 5% acetic acid
    • Slide warmer set at 28-30°C



    • Use the working solution immediately as it lasts only 10 min.
    • Expose sections to the solution for 6 minutes in a dark location at 28-30°C , such as under an aluminum cover. You needn’t work in absolute darkness, but the background decreases slightly if the intensification occurs away from bright light.
    • Stop the reaction with a brief dip in 5% acetic acid.
    • Wash for 10 minutes in dH2O and dry with an air canister or filtered air gun.
    • Cover slip in a medium of your choice.

  6. Imaging
      Equipment & Software

    • Leica DMR Microscope
    • Pyramid PS-26K regulated power supply; 100W Halogen lamp
    • Image tiling stage; Märzhäuser Scan 100 x 100 stage
    • Monochrome camera, 8-bit mode: QImaging QICAM IEEE-1394 camera
    • Tiling acquisition software: Syncroscopy 5.0
    • Image storage system: LaCie 500Gb IEEE-1394 drive


  7. Mosiacking and Registering

    • ir-tools
    • ir-tweak
    • Adobe® Photoshop® CS3


      Mosaic Procedure

    1. Save the Syncroscan workspace (*.sws & the folder of *.bmp files) with the standard naming convention & directory.
    2. Copy the data to the build machine (
    3. Log on to the build machine & open a console
    4. Enter: python/users/shared/ir-tools/ /Users/Shared/LM/yourfolder
    5. Pace around the lab for 5-30 minutes depending on the size of the data. Go tell Robert how bored you are.
    6. The finished mosaics have the extension .png and are placed in the slide directory
      Registration Procedure

    1. Open ir-tweak
    2. Choose a fixed image file
    3. Choose a moving image file
    4. Select control points
    5. Warp until matched
    6. Save moving image registered image
    7. Choose a new moving image file & repeat until all pairs are aligned
    8. Export as Adobe® *_master.psd alpha channels

  8. Image Analysis


      1. Making rgb triplets

    Comment: Silver images are density mapped. To generate “fluorescence-like” images, silver images must be inverted to brightness mapped modes. This is discussed extensivley in Marc and Cameron, 2003.

    1. Open the *_master.psd
    2. Turn on history logging
    3. Copy the desired alpha channels into rgb channels
    4. Invert all rgb channels
    5. Select the rgb channels (cmd ~)
    6. Select all (cmd a) & copy (cmd c)
    7. Create a new rgb file (cmd n)
    8. Paste the rgb triplet (cmd v) and flatten (cmd e)
    9. Save the triplet as *_abc.tif where abc=signal triplet, e.g. yge.


      Isodata Cluster Analysis (pixel-based)

    1. Convert *_master.psd into N inverted monochrome *.tif files
    2. Import as N channels in into PCI Geomatica ImageWorks
    3. Export as an N-channel PCI Geomatica *.pix file with blank channels for theme maps
    4. Open PCI Geomatica Xspace interface & select the Multispectral Analysis Package
    5. Select the ISOCLUS application
    6. Parameter settings
      • FILE: your *.pix file
      • DBIC: the channels containing the monochrome images to be clustered
      • DBOC: the channel to which the theme map should be written
      • NUMCLUS: the estimated number of clusters (usually 15-40)
      • MAXCLUS: the upper bound of estimated clusters (usually 20-50)
      • MINCLUS: the lower bound of estimated clusters (usually 5-10)
      • SEEDFILE: An optional initialization. Default = N-space diagonalization
      • MAXITER: how many iterations to run if convergence fails (about 40)
      • MOVETHRS: the fraction of pixel class changes triggering a new iteration (usually 0.01)
      • SIGGEN: Default NO; set YES if you wish to use SIGSEP
      • SAMPRM: Minimum sample fraction pixels for a class (usually 10 percent)
      • STDEV: Sample std dev triggering a split (usually 10 percent)
      • LUMP: Class center distance triggering a lump (usually 1%)
      • MAXPAIR: Maximum pairs of classes to lump (depends on initial conditions, usually 5)
      • BACKVAL: do not set
      • NSAM: Number of pixel values to sample. For a typical single frame image, set to 1500000.
      • Report: directory path and *.txt filename to write the summary report
    7. Run
    8. File/Open *.pix in ImageWorks and load the theme map channels
    9. File/Open idlpct.pix
    10. File/Load the PCT segment: IDL
    11. File/Save the PCT segment
    12. File/Save *.pix
    13. File/Utility to open the Utility widget
    14. In the Utility Widget: File/Export to…
    15. Select: source as *.pix
    16. Select: Destination file as *.bmp
    17. Select: output format as BMP
    18. Select: source as theme images and add to the export box
    19. Select: source as PCT segments and add to the export box
    20. Select: export
    21. Open Adobe PS, import and save as an indexed tif.


  9. Histogramming: See Cellkit