ANATOMICAL METHODS
1 - ANTIBODY STAINING METHODS
1.1 Mounting animals for observation with Nomarski DIC optics by Monica Driscoll
1.2 Freeze-crack and staining protocol by Janet Duerr
1.3 FMRFamide staining protocol by Chris Li
1.4 Gonad-Intestine staining protocol by Barth Grant
1.5 Antibody staining of formaldehyde-fixed animals by Gary Ruvkun and Michael Finney
1.6 Protocol for staining early embryos by Bruce Bowerman
1.7 Formaldehyde fixation and cytokeletal staining by Raffi V. Aroian
2 - OTHER STAINING METHODS
2.1 FITC (fluorescein isothiocyanate) staining of amphid (ADF, ASH, ASI, ASJ, ASK, ADL) and phasmid (PHA and PHB) neurons :
A stock dye solution containing 20 mg/ml 5-fluorescein isothiocyanate in dimethylformamide can be stored at -20oC. indefinitely. 50 ul of this stock solution is then mixed with 200 ul of M9 buffer and applied evenly to the surface of a 10 ml NGM plate preseeded with a lawn of E. coli. After 2 hr to allow the dye to diffuse into the agar, (final concentration 0.1 mg/ml), live animals are transferred to the plate. After staining for 2 hr to overnight, the animals are transferred to a plate devoid of the dye for at least 10 min to remove free FITC from the intestine before viewing under the microscope (Hedgecock et al, 1985)
2.2 DiI staining of amphid (ASI, ADL, ASK, AWB, ASH, ASJ) and phasmid (PHA and PHB) neurons : See DiI staining of amphids in D-V view; DiI staining of amphids in lat view; DiI staining of phasmids in D-V view .
Note: To stain IL2's with DiI, wash the worm plate with 1ml H2O/50 mM Calcium Acetate and add 50 mM Calcium Acetate into DiI staining solution. At the end of staining, wash with H2O.
2.3 DiO staining of amphid (ASI, ADL, ASK, AWB, ASH, ASJ) and phasmid (PHA and PHB) neurons .
2.4 DAPI (4'-6-Diamidino-2-phenylindole) staining of nuclei: See example image
3- ELECTRON MICROSCOPY TECHNIQUES
3.2 Electron Microscopy Methods
3.2.1Conventional two-step fixation by David H. Hall
3.1.2 High pressure freeze fixation (HPF) by David H. Hall
3.1.3 Microwave aldehyde fixation followed by normal osmium fixation by David H. Hall
3.1.4 Osmium and aldehyde in one-step fixation by David H. Hall
3.1.5 Osmium-only fixation by David H. Hall
3.1.6 Metal mirror(slam-freeze) fixation (MMF) by David H. Hall
3.1.7 Flat Embedding of Worms (Slam Freezing) by Steve Fields (Rand Lab)
3.1.8 SEM preparation of worms by David Greenstein
3.1.9 Laser Hole Fixation by Carolyn Norris (Hedgecock Lab)
3.2 Experimental EM Methods
3.2.1 Freeze substitution in 1% Osmium by Robby Weimer -pdf file
3.2.2 Freeze substitution with tannic acid/osmium_long incubation by Robby Weimer-pdf file
3.2.3 Freeze substitution with tannic acid/osmium_short incubation by Robby Weimer-pdf file
3.2.4 High pressure freezing/freeze-substitution of C. elegans embryos and L1 worms by Richard Fetter
3.2.5 Freeze substitution in Potassium Permanganate by Robby Weimer-pdf file
