Biotinylated donkey anti rabbit igg

Tissue sections were immunostained as previously described [28 (link)]. Primary antibodies included rabbit anti-Ki67 (Thermo Fisher Scientific, Inc., Fremont, CA; Cat. No. RM-9106-S1) (1:200), mouse anti-BrdU (Thermo; Cat. No. MS-1058-PO) (1:250), goat anti-cryptdin related sequence 4C (CRS4C) (gift from Dr. A. J. Ouellette, University of Southern California, Los Angeles, CA) [32 (link)] (1:2000), and rabbit anti-MUC2 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA; Cat. No. sc15334) (1:100). Secondary antibodies included biotinylated donkey anti-rabbit IgG, donkey anti-goat IgG, and donkey anti-mouse IgG (all from Vector Labs, Burlingame, CA). Biotinylated antibodies were linked to avidin-horseradish peroxidase conjugates (Vector Labs), visualized using 3,3′-diamino benzidine (Sigma) for 2 to 5 min, and lightly counterstained with hematoxylin.

Dexmedetomidine was obtained from the Jiangsu HengRui Pharmaceutical Co. Ltd. (Jiangsu, China). Rabbit polyclonal anti-c-Fos antibody was purchased from Abcam (Cambridge, MA, United States). Biotinylated donkey anti-rabbit IgG and avidin-biotin-peroxidase were purchased from Vector Laboratories (Burlingame, CA, United States). Finally, 3, 3-diaminobenzidine-tetra-hydrochloride (DAB) was purchased from Sigma-Aldrich (St. Louis, MO, United States). Dexmedetomidine was dissolved in sterile saline before use.

Animals were dissected in PBS and fixed for 30 min with 4% (wt/vol) Paraformaldehyde in PBS. The following antisera were used for Immunohistochemical analysis: Chicken anti-GFP (Aves, 1:500), Rabbit anti-phospho Chk1 (CST, 1:200), Rabbit anti-phospho Smad (CST, 1:150) Rabbit anti-pH3 (Millipore, 1:500), and Alexa 488/568/647-conjugated Donkey anti-Chicken/Rabbit/Mouse secondary antibodies (Invitrogen, 1:200). Tyramide signal amplification was used as per manufacturer recommendations for p-Chk1 detection. The following reagents were used as part of this protocol: Tyramide amplification buffer and Tyramide reagent (Thermofisher), Vectastain A and B and Biotinylated donkey anti Rabbit IgG (1:200, Vector Labs). Tracheal preparations were flat-mounted in ProLong Diamond Antifade Mountant with DAPI (Molecular Probes) and imaged on Zeiss LSM-780 laser-scanning confocal microscopes. Images were processed using Image J. For quantification of cell number, fixed specimens were mounted in ProLong Diamond Antifade Mountant with DAPI and the number of nuclei were counted on an Olympus BX 53 microscope. The DT of the second thoracic metamere was identified morphologically based on the cuticular banding pattern at anterior and posterior junctions.