Pigs were euthanized by intravenous injection of pentobarbital (150 mg/kg bodyweight) and subsequent exsanguination. Skin tissue at infection sites were extensively excised with a scalpel and scissors, including all layers of the skin, the fascia and the first layer of muscle. The samples were transferred immediately into 10% neutral-buffered formalin solution (approx. 4% formaldehyde; HT501128-4L, Sigma-Aldrich, USA). Additionally to the infected skin tissue a total of 16 lymph nodes were excised. The three main draining lymph nodes (Nll. cervicales superficiales ventrales and dorsales) and the Nll. Subiliaci [20 ] were excised bilaterally from each pig, except for pig number two, for which only the Nll. cervicales superficiales dorsales and Nll. subiliaci were collected.
Ht501128 4l
Manufactured by Merck Group
Sourced in United States
The HT501128-4L is a laboratory equipment product from Merck Group. It is a 4-liter capacity container designed for use in research and scientific applications. The core function of this product is to provide a reliable and durable storage solution for various laboratory materials and samples.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey imaging system, by LI COR (2 mentions)
Infinite 2000 pro plate reader, by Tecan (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Fluoromount g mounting medium with dapi, by Thermo Fisher Scientific (1 mentions)
Ab154856, by Abcam (1 mentions)
Alexa fluor 647 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Axioimager d1 fluorescent microscope, by Zeiss (1 mentions)
Vero e6 cell, by American Type Culture Collection (1 mentions)
Sars cov 2 usa wa1 2020, by BEI Resources (1 mentions)
Agarose 2, by Merck Group (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti ki67, by Novus Biologicals (1 mentions)
A31572, by Thermo Fisher Scientific (1 mentions)
Dabco, by Carl Roth (1 mentions)
P1379 100ml, by Merck Group (1 mentions)
Immunocal, by StatLab (1 mentions)
Dm 2500b microscope, by Leica (1 mentions)
Evos imaging system, by Thermo Fisher Scientific (1 mentions)
Serum free dmem, by Thermo Fisher Scientific (1 mentions)
Oil red o solution, by Merck Group (1 mentions)
27 protocols using ht501128 4l
1
Skin and Lymph Node Collection
2
Quantitative Analysis of Liver Organelles
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Liver tissues were fixed in neutral buffered 10% formalin solution (Sigma-Aldrich, HT501128-4L) embedded in paraffin and cut into 5-μm sections. Liver sections were deparaffinized with Histo-Clear I solution (Electron Microscopy Sciences, 64110-01) and hydrated through decreasing concentration of alcohol solutions. For catalase (peroxisomal marker) and VDAC1/2 (mitochondrial marker) staining, sections were unmasked 20 minutes at 600 W in a microwave with citrate buffer, pH 6.0. Samples were then blocked for 10 min with 3% H2O2 followed by 30 min with 2.5% normal goat serum. Sections were further incubated overnight at 4°C with primary antibodies (1:100 dilution) (catalase ref: ab16731; VDAC1/2, ab154856, Abcam) followed by 30 minutes of incubation with goat anti-Rabbit IgG Alexa Fluor™ 647 (for catalase staining) or goat anti-Rabbit IgG Alexa Fluor™ 488 (for VDAC1/2 staining) (1:200 dilution) (Thermo Fisher Scientific). Samples were mounted with Fluoromount-G mounting medium with DAPI (Invitrogen). All images were captured using a Carl Zeiss Axioimager D1 fluorescent microscope and quantified for number and size of peroxisome and mitochondria using Image J software.
3
Immunofluorescence Staining of Tissue Samples
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The largest section of patient TEs (5–25 sq cm) was fixed in 10% neutral buffered formalin (Sigma Aldrich; HT501128-4L), shaken overnight at 4°C, washed with 1× PBS, and stored in 1× PBS at 4°C, as previously described (25 (link), 93 (link)). Fixed samples from each patient were divided into smaller sections for immunofluorescence staining of bacteria identified via culture-based and microbiome results. Briefly, TE sections were incubated in blocking buffer (1.5% bovine serum albumin and 0.1% sodium azide in 1× PBS) overnight at 4°C and then washed with PBS-T (0.05% Tween-20 in 1× PBS). Primary antibodies (Table S2) were diluted ~1:500 in dilution buffer (0.05% Tween, 0.1% bovine serum albumin, and 0.5% methyl alpha-D-mannopyranoside in 1× PBS) and incubated with the TEs for 2 hours. Following incubation, TEs were washed with PBS-T and incubated with secondary antibodies (Table 2 ) diluted 1:10,000 in dilution buffer for 1 hour. TEs were washed again with PBS-T (three times for 5 minutes) and air dried for >48 hours. The Odyssey Imaging System (LI-COR Biosciences; Cat# B446) was used to detect infrared signal. Negative controls included a small piece of each TE that was treated identically as described above; however, the primary antibodies were excluded.
4
SARS-CoV-2 Variant Growth Kinetics
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SARS-CoV-2 USA-WA-1/2020 (NR-52281) (WA-1), B.1.1.7/Alpha (NR-54000), and B.1.351/Beta (NR-54008) strains were obtained from BEI Resources, and SARS-CoV-2 Delta variant B.1.617.2 hCoV-19/USA/WV-WVU-WV118685/2021 (GISAID Accession ID: EPI_ISL_1742834) was obtained from a patient sample at WVU. These strains were propagated in Vero E6 cells (ATCC-CRL-1586) as described (26 (link), 55 (link)). Vero E6 cells for viral titrations (6-well plate, 106 cells/well) were infected with 10-fold serial dilutions of SARS-CoV-2. At 72 h post infection, cells were fixed overnight with 10% formalin (Sigma HT501128-4L), permeabilized and immunostained with 1 μg/ml of a SARS-CoV cross-reactive N protein antibody 1C7C7, kindly provided by Dr. Thomas Moran at the Icahn School of Medicine at Mount Sinai. For viral growth kinetics, Vero E6 cells (6-well plate, 106 cells/well, triplicates) were infected (multiplicity of infection, MOI 0.01) with SARS-CoV-2 WA-1, Alpha or Beta. At the indicated times after viral infection (12, 24, 48 and 72 h), tissue culture samples were collected and titrated by plaque assay as described (26 (link)).
5
Tissue Sampling and Processing for PNA Analysis
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Blood sample collection was performed at multiple timepoints post-electrotransfer, as indicated in the figures. Blood was processed to serum and stored at −20 °C for further analysis.
For the PNA studies in mice, TA sites were injected with 20 μL of PNA-labeled pDNA at 0.333 mg/mL, immediately after which the animals were euthanized and the target tissue was excised. Extracted tissues were fixed in 10% paraformaldehyde (HT501128-4L, Sigma-Aldrich, St. Louis, MO, USA) for approximately 72 h, after which they were rinsed with 1x PBS (BM-220, Boston Bioproducts, Milford, MA, USA) and securely stored in 1x PBS in light-resistant containers.
For processing, each sample was placed in a potting mold. A 4% w/v Agarose II (A9918-100G, Sigma-Aldrich, St. Louis, MO, USA) gel was evenly poured over each sample and allowed to set at room temperature for an hour. Agarose blocks were sliced into 0.5–1.0 mm sections and then stored in 1x PBS. Sections were further processed by HistoWiz (Queens, NY, USA) for embedding, sectioning, staining, and imaging. Briefly, samples were paraffin wax-embedded, sectioned to 4 μm, mounted on slides, counterstained with DAPI, and subjected to imaging.
For the PNA studies in mice, TA sites were injected with 20 μL of PNA-labeled pDNA at 0.333 mg/mL, immediately after which the animals were euthanized and the target tissue was excised. Extracted tissues were fixed in 10% paraformaldehyde (HT501128-4L, Sigma-Aldrich, St. Louis, MO, USA) for approximately 72 h, after which they were rinsed with 1x PBS (BM-220, Boston Bioproducts, Milford, MA, USA) and securely stored in 1x PBS in light-resistant containers.
For processing, each sample was placed in a potting mold. A 4% w/v Agarose II (A9918-100G, Sigma-Aldrich, St. Louis, MO, USA) gel was evenly poured over each sample and allowed to set at room temperature for an hour. Agarose blocks were sliced into 0.5–1.0 mm sections and then stored in 1x PBS. Sections were further processed by HistoWiz (Queens, NY, USA) for embedding, sectioning, staining, and imaging. Briefly, samples were paraffin wax-embedded, sectioned to 4 μm, mounted on slides, counterstained with DAPI, and subjected to imaging.
6
Immunohistochemical Analysis of Xenograft Tumors
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Harvested xenograft tumors of MDA-MB-436 cells transduced with shCtrl or shMEPCE were fixed using 10% formalin (Sigma cat: HT501128-4L) and embedded in paraffin sectioning. Sectioned slides were rehydrated utilizing a sequence of washes in Xylene, 100%, 95% ethanol, 90%, 80%, 70% ethanol, distilled H2O and finally PBS. Antigen retrieval was performed by placing the slides in a pressure-cooking containing 10 mM sodium citrate buffer (pH 6.0) for 30 min. After cooling to room temperature, slides were incubated in a blocking solution for 2 h followed by overnight incubation with primary antibodies diluted in blocking solution at 4°C in a humidifying chamber. Following a series of washes with PBST, slides were stained with secondary antibodies at room temperature for 2 h at a concentration of 1:500 while protected from light. Following several washes with PBST, coverslips were counterstained with DAPI and sealed with cover glass using MOWIOL. Slides were allowed to dry overnight before visualization and analysis. Primary antibodies used for immunohistochemistry were anti-Ki67 (1:200) (Novus Biological cat: NB500-170) and anti-Cleaved Caspase-3 (1:500) (Cell Signaling cat: 9664). All immunocytochemistry images were taken using a Lecia DM4000 B fluorescence microscope using 100X magnification. Five xenograft sections in at least five 40× fields were visualized and scored.
7
Immunofluorescence Staining of Cryosections
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Cryosections were dried for 10 min at room temperature, fixed for 10 min in 10% formalin (#HT501128-4L, Sigma Aldrich, Germany). Embedding medium was removed with 0.02% Tween20 (T; #P1379-100ML, Sigma Aldrich, Germany) in tris buffered saline (TBS) twice for 5 min. All unspecific binding sites were blocked for 1 h at room temperature in 10% normal donkey serum (#S30-100ML, EMD Milipore, Germany), 1% bovine serum albumin (#0163.2, Roth, Germany) in TBST, followed by overnight incubation of the 1st antibody diluted as indicated in 1% bovine serum albumin in TBST at 4 °C. After 30 min at room temperature 1st antibody was carefully washed away with 4 repetitions of 5 min TBST. The 2nd antibody (A21247; A31571; A21434; A31572; A21432; Invitrogen and 703-545-155; Jackson ImmunoResearch) was diluted 1:500 in 1% bovine serum albumin in TBS, DAPI (300 nM, #D9542-5MG, Sigma Aldrich, Germany) was added, and after 1 h incubation at room temperature and 4 times 5 min wash in TBS, all sections were mounted with Dabco (#0718.1, Roth, Germany) in Mowiol (#0713.1 Roth, Germany). TUNEL staining (In Situ Cell Death Detection Kit, TMR red; #12156792910 Roche; Merck Germany) was applied according to the manual after 2nd antibody and before mounting with Dabco/Mowiol.
8
Histological Analysis of Lung Fibrosis
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Lung tissue sections were fixed in 10% neutral-buffered formalin (Sigma-Aldrich; HT501128-4L) for 24 hours for histologic analysis. Fixed lungs were paraffin embedded and sectioned (5 μm thick), stained with hematoxylin and eosin to examine gross morphology and Masson trichrome stain to visualize collagen deposition, and examined by microscopy. Lung fibrosis was measured using quantitative histology using the Ashcroft method of analysis. All measurements were performed by two independent graders (N.C., A.C.) in a blinded manner. Images were acquired with the Nikon Eclipse E800 microscope (Minato City, Tokyo, Japan) with an Olympus DP70 camera (OM Digital Solutions, Hachioji, Tokyo, Japan). Adjacent 2× images of the lung were stitched together using Adobe Photoshop CS6 (San Jose, CA).
9
Cell Proliferation Assay with Crystal Violet
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Twenty-four hours after transfection, cells were trypsinized and split into 5 individual wells of five separate 12-well plates. Upon adherence, cells were fixed using 10% neutral buffered formalin solution (Sigma-Aldrich, HT501128-4L) and labeled as day 0. Subsequently, the remaining plates were fixed daily from days 2 to 5 (excluding day 1) before staining with crystal violet (Sigma-Aldrich, C0775-100G) for 3 to 5 min at room temperature. Stained wells were washed three times with Milli-Q water and left to dry. Crystal violet stain was solubilized using 10% acetic acid (Sigma-Aldrich, A6283-2.5L). The plates were left on a shaker at room temperature for at least 20 min. The absorbance reading for each well was measured at 595 nm using the Tecan Infinite 2000 PRO plate reader (Tecan, Seestrasse, Switzerland).
10
Encapsulated Cell Electrostimulation in Mice
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Wild-type 8-week-old male C57BL/6J mice were injected with encapsulated engineered cells. All stimulated and non-stimulated mice containing two electrodes were killed after 30 min or 48 h of electrostimulation with DC 4.5 V for 10 s and stored in 10% formalin solution (cat. no. HT501128-4L, Sigma-Aldrich).
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