Protein was extracted as recently described (Zwischenberger et al., 2020). Protein isolates (5–10 μg) were resolved by SDS-PAGE electrophoresis (Bio-Rad Mini PROTEAN Tetra system) using TGX stain-free gradient (4–15%) gels, total protein was visualized using stain-free technology (ChemiDoc MP imaging system), and proteins were electrophoretically transferred (Bio-Rad Trans-blot Turbo Transfer System) to polyvinylidenedifluoride (pvdf) membranes. The membranes were blocked for 1 hr in 3% non-fat dry milk at room temperature, and incubated in primary antibody in 1% non-fat dry milk overnight at 4°C. HRP-linked secondary antibody incubation (Santa Cruz #SC-2004, 1:10,000 dilution) was conducted for 1 hr at room temperature, and the reaction was detected by chemiluminescence (Bio-Rad Clarity Western ECL Substrate). Primary antibodies were as follows: anti-PAI-1 antibody (Abcam ab182973, 1:5,000), anti-CD45 (Abcam, ab10558, 1:1,000), anti-CD11b (Abcam ab133357, 1:1,000), anti-CD3 (Abcam ab16669, 1:500), anti-CD31 (Invitrogen PA5-16301, 1:500), anti-CD34 (Invitrogen PA5-89536, 1:500). Densitometry analysis was performed on the resulting blots using Image Lab software, and normalized by total protein analysis.
Pa5 16301
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PA5-16301 is a laboratory equipment product. It is a core component used in various scientific applications. The details of its specific function and intended use are not available in this factual and unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
Ab133357, by Abcam (1 mentions)
Ab16669, by Thermo Fisher Scientific (1 mentions)
Ab182973, by Abcam (1 mentions)
Ab10558, by Abcam (1 mentions)
Mini protean tetra system, by Bio-Rad (1 mentions)
Mp imaging system, by Bio-Rad (1 mentions)
Sc 2004, by Santa Cruz Biotechnology (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Ab20920, by Abcam (1 mentions)
Ma5 14520, by Thermo Fisher Scientific (1 mentions)
Ab68538, by Abcam (1 mentions)
Ab5694, by Abcam (1 mentions)
Aperio versa, by Leica (1 mentions)
Paraplast, by Leica (1 mentions)
Mouse anti zo 1, by Thermo Fisher Scientific (1 mentions)
Ma5 11315, by Thermo Fisher Scientific (1 mentions)
Rabbit anti cd31, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Sc 133256, by Santa Cruz Biotechnology (1 mentions)
6 protocols using pa5 16301
1
Western Blot Analysis of Immune Markers
2
Histological and Immunohistochemical Analysis of Tissue Scaffolds
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Samples of the scaffolds were fixed in 4% paraformaldehyde for 24 h and stored at 4 °C in 70% ethanol, dehydrated, and embedded in Paraplast (Leica Biosystems). For morphological analysis, 5 μm sections were stained with Hematoxylin–Eosin (H&E) and Masson’s trichrome. Stained tissue sections were scanned at × 40 equivalent resolution using a slide scanner Aperio Versa (Leica Biosystems) and images were captured with Aperio ImageScope 12.4.6 software. Immunohistochemistry was performed according to previously published protocols69 using rabbit polyclonal anti-CD31 (PA5-16301 Invitrogen), 1:50 dilution; rabbit polyclonal anti-α-SMA (ab5694 Abcam), 1:1000 dilution; rabbit monoclonal anti-ki67 (MA5-14520 Invitrogen), 1:50 dilution. Bacterial visualization was performed using rabbit polyclonal anti-Pseudomonas (Abcam ab68538), 1:1000 dilution; rabbit polyclonal anti-Staphylococcus (Abcam ab20920), 1:1000 dilution; and rabbit polyclonal anti-Enterococcus (Abcam ab19980), 1:1000 dilution.
3
Immunofluorescent Staining of Tight Junction Proteins
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Cells seeded on Labtek or transwell were washed with PBS, fixed with cold methanol, permeated with 0.01% Triton (Sigma, St. Louis, MO, USA) solution, blocked with 3% BSA and incubated with the following primary antibodies: rabbit anti-CD31
(1:50, PA516301 Invitrogen), mouse anti-GLUT1 (1:100, MA5-11315 Invitrogen), mouse anti-OCLN (1:50, sc-133256 Santa Cruz Biotechnologies), mouse anti-CLN5 (1:20, 35-2500 Invitrogen), mouse anti-ZO1 (1:50, 33-9100 Invitrogen). Subsequently, cells were incubated with secondary antibodies: Alexa-555 anti-rabbit (1:500, A21429 Invitrogen)
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which this version posted April 14, 2022. ; https://doi.org/10.1101/2022.04.14.488066 doi: bioRxiv preprint and Alexa-488 anti-mouse (1:500, A11029 Invitrogen), respectively. Phalloidin and DAPI staining were used for actin and nuclei visualization, respectively. Images were acquired with a confocal microscope (Carl Zeiss 710) using the image acquisition program Zen (Carl Zeiss). Images were processed and analyzed using ImageJ software (NIH).
(1:50, PA516301 Invitrogen), mouse anti-GLUT1 (1:100, MA5-11315 Invitrogen), mouse anti-OCLN (1:50, sc-133256 Santa Cruz Biotechnologies), mouse anti-CLN5 (1:20, 35-2500 Invitrogen), mouse anti-ZO1 (1:50, 33-9100 Invitrogen). Subsequently, cells were incubated with secondary antibodies: Alexa-555 anti-rabbit (1:500, A21429 Invitrogen)
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which this version posted April 14, 2022. ; https://doi.org/10.1101/2022.04.14.488066 doi: bioRxiv preprint and Alexa-488 anti-mouse (1:500, A11029 Invitrogen), respectively. Phalloidin and DAPI staining were used for actin and nuclei visualization, respectively. Images were acquired with a confocal microscope (Carl Zeiss 710) using the image acquisition program Zen (Carl Zeiss). Images were processed and analyzed using ImageJ software (NIH).
4
Immunohistochemical Characterization of Tissue Samples
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Wax sections were de-waxed in a series of xylene, graded xylene/ethanol mixture, graded ethanol mixture, and finally incubation in PBS. The sections were stained with HE and Masson’s trichrome. Cryosections were mounted on silane-coated slides, air dried, and blocking with 5% bovine serum albumin (BSA) + 0.2% Triton X-100 in PBS if needed. Sections were then left to incubate with primary antibodies overnight at 4 °C, or 4 h at room temperature before overnight incubation at 4 °C. The primary antibody used was rabbit anti-laminin (1:200, L9393, Sigma, St. Louis, MO, USA), rabbit anti-CD31 (1:50, PA5-16301, Thermo Fisher Scientific), rabbit anti-Phospho-ERK1/ERK2 (1:50, 44-680G, Thermo Fisher Scientific, Waltham, MA, USA), and rabbit anti-GluR1 (SD2010) (1:100, MA5-32344, Thermo Fisher Scientific). The secondary antibody for fluorescent photography was CF™ 488 goat Anti-Rabbit IgG (H+L) used (1:400, SAB4600045, Sigma). Primary antibody omission controls were used for all immunostaining protocols to control nonspecific binding. Fluorescent was performed with a Zeiss Axioscope microscope equipped with charge-coupled device (CCD) camera with appropriate filter sets.
5
Western Blotting for Cell Marker Analysis
6
EdU Labeling and Immunostaining in Tissue
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Animals were treated with 50 mg/kg EdU i.p. (BCK488-IV-IM-S, baseclick GmbH) 4 h prior to euthanasia. Animals were cardially perfused with 4.5% phosphate-buffered formaldehyde solution (Roti-Histofix 4.5%, 2213, Carl Roth). Tissue was frozen after cryoprotection with 30% sucrose solution and 10 µm cryosections were cut. The sections were permeabilized with 0.5% Triton X-100 in PBS for 20 min. The EdU Click Kit (BCK488-IV-IM-S, baseclick GmbH) was used for EdU detection. After washing, sections were incubated with the reaction cocktail for 30 min as per the manufacturer’s instructions. Afterwards sections were incubated with anti-nestin (1:400, ab6320, Abcam, RRID:AB_308832) and anti-CD31 (1:50; PA5-16301, ThermoFisher Scientific, RRID:AB_10981955) antibodies.Goat anti-rabbit Alexa Fluor 594 (1:400; A-11037, ThermoFisher Scientific, RRID:AB_2534095) and goat anti-mouse Alexa Fluor 633 (1:400; A-21052, ThermoFisher Scientific, RRID:AB_2535719) secondary antibodies were used. Images were acquired using a Leica TCS SP5 confocal microscope (LAS software version 2.7.3.9723).
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