MISFISHIE FOR ZEBRAFISH WHOLE MOUNT IN SITU HYBRIDIZATIONS

 

Experiment Design:

 

Type of experiment:  Whole-mount in situ hybridization analysis of expression patterns of gene transcripts in wild type zebrafish embryos.

            ►Experimental Factors:  Different cDNA samples and developmental stages.

The number of hybridization/stain performed in the experiment: one hybridization with gene specific DIG-labeled RNA probes, one binding reaction of anti-DIG antibody conjugated with alkaline-phosphotase and one round of alkaline-phosphotase staining.

URL of any supplemental websites or database: http://zfblasta.tch.harvard.edu/stemcell/

Contact information for the experiments: Yi Zhou at yzhou@enders.tch.harvard.edu and Leonard I. Zon at zon@enders.tch.harvard.edu

 

Speciments (BioMaterial) used, section preparation (treatment):

 

The origin of the biological sample: Danio rerio (zebrafish), gender unknown. Developmental stages are at 12, 24, 36, 72 hours post fertilization.

Manipulation of biological samples into sections and protocols used:  embryos at different stage of development were de-chorionated and fixed in 4% paraformaldhyde overnight and then stored in 100% methanol.  Embryos were rehydrated before performing in situ hybridization experiments.  Please see the whole mount in situ protocol at http://zfblasta.tch.harvard.edu/stemcell/?

 

Probe or antibody information:

 

Protocol for how the probes or antibodies were designed and produced or from where they were obtained: DIG-labeled RNA probes were derived from cDNAs of adult zebrafish kidney library using a protocol described in the whole-mount in situ hybridization protocol (http://zfblasta.tch.harvard.edu/stemcell/).   These cDNAs were cloned into Lamda ZAP Express expression vector system (Stratagen) and later in vivo excised into pBS-CMV plasmid vector (Stratagen).  The directional cloning sites are NotI-XhoI.  Sequence information of a particular probe can be found by search the database at http://zfblasta.tch.harvard.edu/stemcell/blast using the clone names as key words.

 

► Unambiguous identification of the probe or antibody, at minimum:

            Gene identifier and the reference database containing the identifier:

Hs: chr16:166,694-167,448+

Mm: chr11:32,182,048-32,182,843+

Dr: chromosome fragment ctg11168: 18060 - 125826

Full sequence of the probe or antibody: Sequence information of a particular probe can be found by searching the sequence database at http://zfblasta.tch.harvard.edu/stemcell/blast using the clone name as a key word.

Staining protocols and parameters:

 

► Number of detectable probes or antibodies on the hybridization or stain: One.

The protocol and conditions used to fabricate the hybridization or stain, including mounting onto the substrate and subsequent treatments: see http://zfblasta.tch.harvard.edu/stemcell/protocol

 

Imaging data and parameters:

The image(s):

Image acquisition parameters:

o       Detection method by which hybridization or staining is observed (for each channel if multiple probes or antibodies are used): Alkaline phosphotase staining.

o       Image scale or total instrumental magnification: whole embryo pictures.  Scales of amplification are not necessary.

o       Imaging hardware and software used: Plain digital pictures.

o       Image editing software used: Adobe Photoshop.

 

Characterizations:

 

·        Define (or choose from an ontology) each structural unit used for classification [Alternate term: “structural component”, “tissue feature”?]. 

Blood cells.  Please see also http://zdb.wehi.edu.au:8282/zf_info/anatomy/dict/sum.html for detailed description of structures.

·        Define (or choose from an ontology) the staining intensity scale.  It is recommended that the three-state scale of “none”, “equivocal”, or “intense” be used, but another scale such as the traditional 0 – 2+ or 0 – 3 scales may be used as long each gradation used is properly defined.  Whole mount in situ staining varies so much and it is not a method for quantifying expression levels.  Normally, no staining intensity scale is defined.

·        For each structural unit in each slide (or optionally each image), provide quantitative measurements or estimates of: NA

Staining intensity (as defined above)

Fraction of the structural unit population exhibiting that intensity

Other optional annotations for the structural unit, e.g., feature density, qualitative characteristics of the structural unit.  Use of referenced-ontology terms is encouraged.