Immediately after clot retrieval, i.e., still within the catheter laboratory, thrombus material was fixed in phosphate-buffered formalin. The formalin-fixed specimens were embedded in paraffin (Leica, Wetzlar, Germany), cross-sectioned at 4-µm thickness and stained with hematoxylin and eosin (H&E), and Martius scarlet blue (MSB) (Atom Scientific, Cheshire, UK). Subsequently, based on H&E staining, thrombi were characterized according to their overall appearance into erythrocytic, layered, and serpentine [11 (link)]. Categorization was done by visual assignment of two independent investigators (Michael K. Schuhmann, Peter Kraft). In case of divergent results after first view, investigators independently re-categorized the clots and finally had to reach an agreement. Additionally, using MSB-stained sections, the content of RBCs and fibrin/collagen was quantitatively determined. Accordingly, thrombi were classified as red (RBCs outnumber fibrin/collagen ≥ 15%) or white (fibrin/collagen outnumber RBCs ≥ 15%). All others were classified as mixed [12 (link)]. To assess for thromboinflammation, all thrombi were stained immunohistochemically for CD4+ T cells (abcam; ab133616, Cambridge, UK), CD68+ monocytes (Acris; AM331235U-N, Herford, Germany) and von Willebrand factor (abcam; ab6994, Cambridge, UK).
Ab6994
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab6994 is a monoclonal antibody that recognizes the human GAPDH protein. GAPDH is a key enzyme involved in glycolysis. The antibody can be used to detect GAPDH expression in various cell and tissue types.
Automatically generated - may contain errors
Lab products found in correlation
Ab15580, by Abcam (6 mentions)
Ab7817, by Abcam (6 mentions)
Triton x 100, by Merck Group (6 mentions)
Ab5694, by Abcam (4 mentions)
Ab24590, by Abcam (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Ab28364, by Abcam (3 mentions)
Ab53554, by Abcam (3 mentions)
Protein block solution, by Agilent Technologies (3 mentions)
A7811, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Saponin, by Merck Group (3 mentions)
Ab955, by Abcam (3 mentions)
Tunel solution, by Roche (3 mentions)
Axio scope a1, by Zeiss (3 mentions)
Hoechst 33342, by Merck Group (3 mentions)
Ab9465, by Abcam (3 mentions)
Ab6721, by Abcam (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Ab177487, by Abcam (2 mentions)
101 protocols using ab6994
1
Thrombus Characterization and Inflammation
2
Uterine Horn Regeneration Assessment
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On days 30 and 90 post-transplantation, a subset of rats (n = 4 uterine horns) from each group was euthanized. The injured site of each uterine horn was dissected, fixed in neutral formaldehyde for 24 h, and embedded in paraffin perpendicularly. Sections (3 μm) of uterine horns were then prepared transversally.
Hematoxylin and eosin (H&E) staining was then employed to observe tissue structure. Sections were stained for immunohistochemistry with anti-α-smooth muscle actin antibody (α-SMA, 1:1500, ab5694, Abcam) and anti-von Willebrand factor antibody (vWF; 1:10000, ab6994, Abcam). The thickness of endometrium was measured using ImageJ (National Institutes of Health, USA). The percentage of α-SMA positive area (α-SMA positive area of the injured region/total α-SMA positive area) was used to evaluate smooth muscle abundance with the Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA). Blood vessel density was evaluated in at least three randomly selected fields per section under a magnification of × 400 [6 (link)].
Hematoxylin and eosin (H&E) staining was then employed to observe tissue structure. Sections were stained for immunohistochemistry with anti-α-smooth muscle actin antibody (α-SMA, 1:1500, ab5694, Abcam) and anti-von Willebrand factor antibody (vWF; 1:10000, ab6994, Abcam). The thickness of endometrium was measured using ImageJ (National Institutes of Health, USA). The percentage of α-SMA positive area (α-SMA positive area of the injured region/total α-SMA positive area) was used to evaluate smooth muscle abundance with the Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA). Blood vessel density was evaluated in at least three randomly selected fields per section under a magnification of × 400 [6 (link)].
3
Validating EV Protein Expression
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Complement component (C2), vascular endothelial growth factor (VEGF), and von Willebrand factor (vWF) proteins, which were all enriched in EVs, were selected for validation, and their expression in EVs and MSCs measured by Western blot (Novus: Cat#NBP1-58985, Santa Cruz Biotech: cat# sc-152, and Abcam: cat#ab6994, respectively). Similarly, we validated the expression of RNA polymerase-associated protein RTF1 homolog (RTF1) and FOS-Like Antigen 1 (FOSL1) excluded from EVs (Abcam: cat#ab99362 and LifeSpan BioSiences, Inc. cat#:aa130-179, respectively).
4
Immunohistochemical Analysis of Lung Tissue
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Parrafin embedded lung tissue sections were stained for fibronectin (Sigma, F3648), vWF (Abcam, ab6994) and reactive oxygen species (ROS, Abcam, ab5512) with negative control isotypes used on serial sections (Dako). Sections were imaged using an Olympus BX60 microscope and densitometry was analysed with ImageJ software.
5
Histological analysis of urethral tissue
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The animals were sacrificed by overdosed anesthetic drugs, and the urethra and surrounding tissue were excised during surgery based on the silk-marked site. The 8 μm slides from paraffin-embedded tissue were stained with HE and Masson's trichromatic staining. ImageJ software was used for quantitative analysis of collagen to calculate the percentage of the positive area (positive area/total tissue area). To assess the inflammatory response, T lymphocytes, B lymphocytes, and macrophages were observed. And a two-step® Poly-HRP anti-mouse/rabbit IgG detection system (PV-6000, ZSGB-BIO) was implemented for the final colorimetric signal development. The OCT-embedded tissue was frozen and sectioned for immunofluorescence detection. Vascular tissue immunofluorescence staining was performed using a rabbit polyclonal antibody against von Willebrand factor (vWF) (ab6994, Abcam, UK). After the cell nuclei were stained with DAPI, immunofluorescent images were acquired by a confocal laser scanning microscopy (CLSM) (Nikon ECLIPSE Ni-U, Nikon Imaging Instruments Sales Co. Ltd.). Similarly, the quantitative analysis of the fluorescence image was performed using ImageJ software.
6
Quantitative Immunohistochemistry of Brain Sections
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Immunohistochemistry of cryo-embedded brain sections was performed using anti-albumin (ab10685, abcam, Cambridge, UK), anti-von Willebrand factor (ab6994, abcam), anti-occludin (611090, BD Transduction Laboratories, San Jose, CA, USA), and anti-claudin 5 (35-2500, Invitrogen, Waltham, MA, USA) antibodies as described recently [22 (link)]. Quantification of the albumin-positive area was carried out from 5 subsequent slices per animal with an interval of 100 µm (5-fold magnification). Claudin 5 and occludin staining were quantified using 40-fold magnification by measuring the length of continuous (undisrupted) junctional lines in three optical fields within the regions of interest, applying Image J software version 1.52j (NIH, Bethestda, MD, USA). DAPI staining was used to counterstain cell nuclei and to quantify infarct volumes. Sections were analyzed under a microscope (Leica DMI 8, Leica Microsystems, Wetzlar, Germany) equipped with a charge-coupled device camera.
7
Quantifying Angiogenesis and Proliferation in Xenograft Tumors
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To estimate angiogenesis and cell proliferation in xenograft tumors, immunofluorescence staining was performed using the rabbit polyclonal anti-von Willebrand factor (vWF at 1:100) (ab6994; Abcam, Cambridge, UK) and mouse monoclonal anti-Ki 67 (1:100) (ab238020; Abcam) as the primary antibodies. The vWF- and Ki 67-positive areas in five random fields (200× magnification) were quantified using ImageJ software (n = 4 in each group). The positive areas were quantified as the ratio of the vWF- or Ki 67-positive area to the total cell expression area.
8
Immunofluorescence Staining of EC and SMC
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Passage 1 EC and SMC were grown to confluence on plastic dishes or on chitosan/PCL membranes. After that, they were fixed with 4% PFA (paraformaldehyde) for 10 min, permeabilized with 0.05% Triton X-100 for 10 min, and blocked with 1% BSA (bovine serum albumin). The cells were stained with primary antibodies overnight at 4 °C, washed with PBS and incubated with secondary antibodies for 1 h at room temperature. The stained cells were analysed with an inverted fluorescence microscope (Nikon Ti-E) using Nikon AR software.
The following primary antibodies were used: anti-human CD31 (M0823, DAKO, 1:50), anti-α-SMA (DAKO, M0851, 1:50), anti-smooth muscle myosin heavy chain 11 (Abcam, ab82541, 1:500), anti-human CD90 (eBioscience, 14090982, 1:100), anti-Von Willebrand factor (Abcam, ab6994, 1:200), anti-fibronectin (Abcam, ab6328, 1:200), anti-elastin (Abcam, ab21610, 1:200), and anti-collagen IV (Life Span, 1:200).
The following secondary antibodies were used: Alexa Fluor 568 goat anti-mouse IgG1 (Life Technologies, A21124, 1:400), Alexa Fluor 488 goat anti-mouse IgG1 (Life Technologies, A21121, 1:400), Alexa Fluor 568 goat anti-mouse IgG2a (Life Technologies, A21134, 1:400), Alexa Fluor 568 goat anti-mouse IgG (H + L) (Life Technologies, A11031, 1:400), and Alexa Fluor 488 goat anti-mouse IgG (H + L) (Life Technologies, A11029, 1:400).
The following primary antibodies were used: anti-human CD31 (M0823, DAKO, 1:50), anti-α-SMA (DAKO, M0851, 1:50), anti-smooth muscle myosin heavy chain 11 (Abcam, ab82541, 1:500), anti-human CD90 (eBioscience, 14090982, 1:100), anti-Von Willebrand factor (Abcam, ab6994, 1:200), anti-fibronectin (Abcam, ab6328, 1:200), anti-elastin (Abcam, ab21610, 1:200), and anti-collagen IV (Life Span, 1:200).
The following secondary antibodies were used: Alexa Fluor 568 goat anti-mouse IgG1 (Life Technologies, A21124, 1:400), Alexa Fluor 488 goat anti-mouse IgG1 (Life Technologies, A21121, 1:400), Alexa Fluor 568 goat anti-mouse IgG2a (Life Technologies, A21134, 1:400), Alexa Fluor 568 goat anti-mouse IgG (H + L) (Life Technologies, A11031, 1:400), and Alexa Fluor 488 goat anti-mouse IgG (H + L) (Life Technologies, A11029, 1:400).
9
Immunohistochemical Analysis of Brain Markers
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Samples were blocked with a solution containing 0.3% Triton, 3% goat serum, and 1% bovine serum albumin. To label BrdU, sections were preincubated with 2N HCL at 37 °C, followed by incubation with rat monoclonal antibody against BrdU (1:400; ab6326; Abcam, UK). For others, sections were incubated with antibody against TREM-1 (1:200; ab217161, Abcam, UK), Iba-1 (1:500; #019-19741, Wako, Japan), CD68 (1:200; MCA1957GA, AbD Serotec, UK), GFAP (1:500; ab4648, Abcam, UK), NeuN (1:500; ab177487, Abcam, UK), MBP (1:500; ab7349, Abcam, UK), vWF (1:500; ab6994, Abcam, UK), MPO (1:200; sc-16128-R, Santa Cruz Biotechnology, USA), SYK (1:200; #13198, Cell Signaling Technology, USA), and Gasdermin D (GSDMD) (1:200; sc-393581, Santa Cruz Biotechnology, UK) overnight at 4 °C. Sections were then incubated with appropriate secondary antibodies and DAPI (Sigma-Aldrich, USA). The positive signal was quantified by Image J software (NIH, USA).
10
Protein Expression Analysis for Angiogenesis
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SPECs and FOS were collected after 1, 2, and 3 days of culture in 2% linear agarose molds as previously described. Samples were snap frozen and mechanically homogenized in RIPA lysis buffer with protease inhibitor cocktail. Samples were maintained in constant agitation for 2 h at 4°C and centrifugated for 20 min at 16,000 g at 4°C. Supernatant was stored in fresh tube at −20°C. Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific 23227) was used to estimate protein concentration for samples as per manufacturer's instructions. Before gel electrophoresis, samples were diluted in RIPA buffer to attain 20 μg of protein in 20 μL solution, and further diluted 1:1 in 2 × Laemmli Sample buffer to attain 40 μL loading volumes. Samples were loaded onto Any kD™ Mini-PROTEAN® TGX™ Precast Protein Gels. Following protein separation and overnight transfer onto PVDF membranes, western blots were performed using antibodies toward GAPDH (loading control) (Calbiochem CB1001; 1:1000), VEGFR2 (Abcam; ab39256, 1:900), VE-Cadherin (ThermoFisher Scientific 36-1900; 1:250), vWF (Abcam; ab6994, 1:500), and DLL4 (Abcam; ab7280, 1:1000).
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