Histopathology was performed as described [18 (link)]. In brief, formalin-fixed paraffin-embedded wound sections were deparaffinized and stained with hematoxylin & eosin (H&E), Mason Trichrome and Gram/Twort stain (Newcomer Supply Inc.), using standard procedures. Immunohistochemical staining of OCT embedded frozen- sections were performed as described earlier [18 (link)] using the following primary antibodies: anti-Pseudomonas antibody (custom developed by Covance, Denver, PA), anti-Acinetobacter antibody (custom developed by Covance, Denver, PA) and ZO-1 and ZO-2 (Invitrogen). Because of the high auto-fluorescence in porcine tissue and weak GFP signal emitted from our PAO1::gfp strain, we chose to visualize P. aeruginosa with anti-Pseudomonas antibody rather than relying on the GFP signal. Mosaic images were collected using a Zeiss Axiovert 200 inverted fluorescence microscope supported by an AxioCam digital camera, a motorized stage, and guided by Axiovision software (Zeiss). Each mosaic image was generated by combining a minimum of 100 images. Confocal Laser Scanning Microscopy (CLSM) was performed using a Fluoview FV1000 spectral confocal microscope (Olympus, Pittsburgh, PA) equipped with an argon laser. Z-stack images were created by merging serial scans of tissue sections (20-50 μm).
Zo 1 and zo 2
Manufactured by Thermo Fisher Scientific
Sourced in Canada
The ZO-1 and ZO-2 are laboratory equipment products from Thermo Fisher Scientific. The ZO-1 is a protein that functions as a tight junction-associated protein, while the ZO-2 is another tight junction-associated protein. These products are intended for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam digital camera, by Zeiss (2 mentions)
Axiovision software, by Zeiss (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Zen 2012 sp1, by Zeiss (1 mentions)
α tubulin, by Abcam (1 mentions)
Alexa fluor conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
E cadherin, by BD (1 mentions)
Cldn3, by Abcam (1 mentions)
Slowfade gold with dapi, by Thermo Fisher Scientific (1 mentions)
Rhoa and cdc42, by Santa Cruz Biotechnology (1 mentions)
2 protocols using zo 1 and zo 2
1
Histopathological Imaging of Wound Infection
2
Immunostaining of Tight Junction Proteins
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Immunostaining was performed on paraffin sections (for α-tubulin), cryosections or whole embryos fixed in 4% PFA or 10% trichloroacetic acid at 4 °C. Samples were incubated overnight at 4 °C with primary antibodies: Cldn1, −4 and −8 (Invitrogen, Carlsbad, USA, 1:25–1:50), Cldn3 (Abcam, Cambridge, UK, 1:50), Cldn14 (Sigma Aldrich, Oakville, Canada, 1:25), ZO-1 and ZO-2 (Invitrogen, 1:50, Carlsbad, USA), anti-disphospho-myosin light chain (Thr18/Ser19) (Cell Signaling, Ipswich, USA, 1:50), RhoA and Cdc42 (Santa Cruz, Santa Cruz, USA, 1:50), Par3 (Millipore, Etobicoke, Canada, 1:250), Dlg1 (US Biologicals, Salem, USA, 1:50), E-cadherin (BD Transduction, San Jose, USA, 1:100), α-tubulin (Abcam, Cambridge, UK, 1:100) or Vangl2 (gift from Dr. M Montcouquiol, 1:500). Alexa Fluor-conjugated secondary antibodies (1:500) were added for 1 h at RT. F-actin was detected using Alexa Fluor-conjugated Phalloidin (Molecular Probes, Eugene, USA). Sections and flat-mounted embryos were coverslipped with SlowFade Gold with DAPI (Molecular Probes, Eugene, USA) and imaged using a Zeiss LSM780 laser scanning confocal microscope. Colocalization and immunoquantification was performed using ZEN 2012 SP1 software (Carl Zeiss Microscopy, Germany).
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