Cryosectioning and In Situ Hybridization of Larval Samples

For cryosections, larvae were processed as described.^{61 (link)} Larvae were cut as 16 μm sections on a Leica CSM1816 cryostat. For in situ hybridization, sections were hybridized as described^{62 (link)} with published antisense probes against collagen 2a1a^{63 (link)} or collagen 10a1a.^{64 (link)} Immunohistochemistry was performed as described^{62 (link)} without the proteinase K step, with mouse anti-PCNA (Proliferating Cell Nuclear Antigen; 1/500; Santa Cruz Biotech; PC10), Alexa Fluor 488 conjugated Donkey anti-mouse secondary antibody (1/100 Jackson Immunoresearch 715-545-150) and DAPI (1:2000 Life Technologies D1306). Fluorescence imaging was conducted on a Leica SP8 confocal microscope with a 63X oil immersion objective.

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Heubel B.P., Bredesen C.A., Schilling T.F, & Le Pabic P. (2020). Endochondral growth zone pattern and activity in the zebrafish pharyngeal skeleton. Developmental dynamics : an official publication of the American Association of Anatomists, 250(1), 74-87.

Publication 2020

CSM1816 cryostat mouse anti-PCNA (Proliferating Cell Nuclear Antigen; Alexa Fluor 488 conjugated Donkey anti-mouse secondary antibody DAPI SP8 confocal microscope

Alexa fluor 488 Anti antibody Collagen Confocal microscope Cryosections Dapi Donkey Immersion Immunohistochemistry In situ hybridization Larvae Mouse Proliferating cell nuclear antigen Proteinase k

Corresponding Organization : University of North Carolina Wilmington

Other organizations : University of Delaware, University of California, Irvine

Top 5 similar protocols

Variable analysis

independent variables

Cryosectioning method: Larvae were cut as 16 μm sections on a Leica CSM1816 cryostat
In situ hybridization: Sections were hybridized with published antisense probes against collagen 2a1a or collagen 10a1a
Immunohistochemistry: Performed without the proteinase K step, with mouse anti-PCNA antibody and Alexa Fluor 488 conjugated Donkey anti-mouse secondary antibody

dependent variables

Collagen 2a1a expression
Collagen 10a1a expression
PCNA expression (as a marker of cell proliferation)

control variables

Larvae processing: Larvae were processed as described in the referenced publication
In situ hybridization: Sections were hybridized as described in the referenced publication
Immunohistochemistry: Performed as described in the referenced publication, without the proteinase K step

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!