The staining protocol of IHC was performed using the two-step method described previously [23 (link)]. A rabbit polyclonal antibody of FOXJ1 [1: 100, Abcam, UK] was used as the primary antibody. The EnVision™+/HRP working solution was provided by DAKO (Agilent Technologies Inc., Glostrup, Denmark). Diaminobenzidine was added to visualize the reaction products and hematoxylin was used for counterstaining. The staining of FOXJ1 was independently scored by 2 pathology experts blinded to the clinical and pathological parameters according to the criteria previously described [23 (link)].
Foxj1
Manufactured by Abcam
Sourced in United Kingdom, Canada
FOXJ1 is a transcription factor that is involved in the regulation of cilia formation and function. It plays a crucial role in the development and maintenance of ciliated cells, which are found in various tissues such as the respiratory system, reproductive system, and central nervous system.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Brachyury, by Abcam (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Amphotericin, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Vimentin, by Agilent Technologies (1 mentions)
Pronase, by Roche (1 mentions)
Lsm 5 pascal, by Zeiss (1 mentions)
Fluorescein isothiocyanate (fitc), by Abcam (1 mentions)
Oct compound, by Thermo Fisher Scientific (1 mentions)
Antifade solution, by Merck Group (1 mentions)
β catenin, by Abcam (1 mentions)
Phycoerythrin pe, by Abcam (1 mentions)
4 protocols using foxj1
1
Immunohistochemical staining of FOXJ1
2
Immunofluorescence Characterization of Stem Cells
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The following primary antibodies were used for fluorescence microscopy experiments at 1:200 dilution and secondary antibodies at 1:400 dilution: WT1 (Abcam, ab89901), PAX8 (Proteintech, 21384-1-AP), TUBB4A (Abcam, ab1315), BRACHYURY (Abcam, ab20680), POU5F1 (Stemgent, 09-0023), Nanog (Stemgent, 09-0020), SOX2 (Stemgent, 09-0024), TRA-1-60 (Stemgent, 09-0010), TRA-1-81 (Stemgent, 09-0011), SSEA4 (Stemgent, 09-0006), CDX2 (Biocare Medical, CM226A), SIX2 (Proteintech, 11562-1-AP), FOXJ1 (Abcam, ab40869), CDH1 (R&D System, AF648), OVGP1 (SIGMA, HPA062205) and DAPI (Molecular Probes, D3571).
3
Isolation and Culture of Airway Cells
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Tissue fragments were washed, minced, and incubated for 48 hours in MEM (Invitrogen, Burlington, Canada) containing 1.4 mg/ml Pronase and 0.1 mg/ml DNase I (Roche Diagnostics, Laval, Canada) at 4°C. Enzymes were inactivated by addition of fetal bovine serum (FBS) to 10% (v/v). Dissociated cells were collected; resuspended in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.625μg amphotericin (Invitrogen); and transferred to a positively charged culture plate for 3 to 4 hours. Nonadherent cells were collected and seeded onto collagen type IV–coated Transwell inserts at >75% confluency, as previously described [14] . All cultures were maintained with culture medium in both chambers. Medium was replaced every 48 hours until cultures were confluent. Cultures consisted of 49.6%±2.9% (mean±SEM) secretory epithelial cells, 38.0%±2.0% ciliated epithelial cells, and 11.7%±1.6% stromal cells, as determined by immunostaining for Pax8 (ProteinTech, Rosemont, IL), FoxJ1 (Abcam, Cambridge, MA), and vimentin (Dako, Mississauga, Canada), respectively (n=4).
4
Immunofluorescent Characterization of Organoid Cultures
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Organoids were embedded in an optimum cutting temperature (OCT) compound (Thermo
Fisher Scientific, MA, USA) and stored at −80oC. The OCT-embedded
organoids were sectioned at 5 μm thickness. The sections were removed from the
OCT, washed with phosphate-buffered saline (PBS), and fixed with 4%
paraformaldehyde for 15 min. The sections were permeabilized with 0.1% Triton
X-100, blocked with 5% FBS, and treated with primary antibodies (1:100, FOXJ1
[ciliated cell marker], 1:100, PAX8 [secretory cell marker], and 1:100,
beta-catenin, all purchased from Abcam) for 1.5 h. The sections were washed with
PBS and 0.1% Tween 20, then treated with secondary antibodies [Fluorescein
isothiocyanate (FITC) or phycoerythrin (PE); Abcam] for 1 h. The sections were
then mounted in antifade solution (Sigma-Aldrich) and washed with PBS plus 0.1%
Tween 20. The nuclei were counterstained with DAPI, and the slides were
subjected to confocal laser-scanning microscopy (LSM5 PASCAL; Carl Zeiss, Jena,
Germany).
Fisher Scientific, MA, USA) and stored at −80oC. The OCT-embedded
organoids were sectioned at 5 μm thickness. The sections were removed from the
OCT, washed with phosphate-buffered saline (PBS), and fixed with 4%
paraformaldehyde for 15 min. The sections were permeabilized with 0.1% Triton
X-100, blocked with 5% FBS, and treated with primary antibodies (1:100, FOXJ1
[ciliated cell marker], 1:100, PAX8 [secretory cell marker], and 1:100,
beta-catenin, all purchased from Abcam) for 1.5 h. The sections were washed with
PBS and 0.1% Tween 20, then treated with secondary antibodies [Fluorescein
isothiocyanate (FITC) or phycoerythrin (PE); Abcam] for 1 h. The sections were
then mounted in antifade solution (Sigma-Aldrich) and washed with PBS plus 0.1%
Tween 20. The nuclei were counterstained with DAPI, and the slides were
subjected to confocal laser-scanning microscopy (LSM5 PASCAL; Carl Zeiss, Jena,
Germany).
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