Immunochemistry for Computational Molecular Phenotyping
- 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.
- Dehydration and Infiltration
- 0.1 M phosphate buffer, pH 7.4 (PB)
- 100% Acetone
- 100% anhydrous MeOH and 75%, 85%, 95% MeOH in dH2O
- Eponate resin
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
- 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
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
- 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.
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.
- Mosiacking and Registering
- Adobe® Photoshop® CS3
- Save the Syncroscan workspace (*.sws & the folder of *.bmp files) with the standard naming convention & directory.
- Copy the data to the build machine (184.108.40.206/Shared/LM/yourfolder).
- Log on to the build machine & open a console
- Enter: python/users/shared/ir-tools/LMBuild.py /Users/Shared/LM/yourfolder
- Pace around the lab for 5-30 minutes depending on the size of the data. Go tell Robert how bored you are.
- The finished mosaics have the extension .png and are placed in the slide directory
- Open ir-tweak
- Choose a fixed image file
- Choose a moving image file
- Select control points
- Warp until matched
- Save moving image registered image
- Choose a new moving image file & repeat until all pairs are aligned
- Export as Adobe® *_master.psd alpha channels
- Image Analysis
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.
- Open the *_master.psd
- Turn on history logging
- Copy the desired alpha channels into rgb channels
- Invert all rgb channels
- Select the rgb channels (cmd ~)
- Select all (cmd a) & copy (cmd c)
- Create a new rgb file (cmd n)
- Paste the rgb triplet (cmd v) and flatten (cmd e)
- Save the triplet as *_abc.tif where abc=signal triplet, e.g. yge.
Isodata Cluster Analysis (pixel-based)
- Convert *_master.psd into N inverted monochrome *.tif files
- Import as N channels in into PCI Geomatica ImageWorks
- Export as an N-channel PCI Geomatica *.pix file with blank channels for theme maps
- Open PCI Geomatica Xspace interface & select the Multispectral Analysis Package
- Select the ISOCLUS application
- 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
- File/Open *.pix in ImageWorks and load the theme map channels
- File/Open idlpct.pix
- File/Load the PCT segment: IDL
- File/Save the PCT segment
- File/Save *.pix
- File/Utility to open the Utility widget
- In the Utility Widget: File/Export to...
- Select: source as *.pix
- Select: Destination file as *.bmp
- Select: output format as BMP
- Select: source as theme images and add to the export box
- Select: source as PCT segments and add to the export box
- Select: export
- Open Adobe PS, import and save as an indexed tif.
- Histogramming: See Cellkit