After sacrifice, mice lungs were perfused with 2% paraformaldehyde followed by paraffin embedding. Paraffin sections were stained with Masson trichrome to determine collagen deposition. Collected human lungs samples were perfused with 2% paraformaldehyde, followed by saturation in 30% sucrose for frozen sections or paraffin embedding. Antibodies against ATF3 (HPA001562, Sigma) were used to perform the immunohistochemistry studies in paraformaldehyde fixed paraffin embedded lung tissue. Immunofluorescence analyses were performed in paraformaldehyde‐fixed sucrose‐saturated frozen sections of lung tissue using antibodies against ATF3 (HPA001562, Sigma) and ABCA3 (WMAB‐ABCA‐17, Seven Hills).
Hpa001562
Manufactured by Merck Group
Sourced in United States
HPA001562 is a laboratory instrument designed for the detection and quantification of specific molecules or analytes within a sample. It utilizes a recognized analytical technique to provide accurate and reliable results. The core function of this product is to assist researchers and scientists in their analytical workflows.
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Lab products found in correlation
Ap0063, by Bioworld Technology (2 mentions)
Axiocam mrm, by Zeiss (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Txnip, by Thermo Fisher Scientific (1 mentions)
Axiovision z1, by Zeiss (1 mentions)
Alexa 555, by Thermo Fisher Scientific (1 mentions)
Immunohistochemical staining kit, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Anti β tubulin antibody, by Merck Group (1 mentions)
Enhanced chemiluminescence detection kit, by Merck Group (1 mentions)
Phosphatase inhibitor cocktails 2 and 3, by Merck Group (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
P5282, by Merck Group (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Confocal microscope, by Leica (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
His tag, by Abmart (1 mentions)
11 protocols using hpa001562
1
Lung Tissue Analysis by Immunohistochemistry
2
Immunostaining of Human and Mouse Corneas
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Three separate human corneas, ages 27, 42, and 62, were used with postmortem delay of no more than 72 hours (Eversight Eye Bank, Chicago, IL, USA). Frozen sections (5 μm) of optimal cutting temperature compound-embedded human and mouse corneas were fixed in 4% paraformaldehyde, blocked in 10% goat serum in PBS and incubated overnight with the following primary antibodies: rabbit polyclonal antibodies against Kl67 (SP6- 1:500 dil; Sigma-Aldrich Corp., St. Louis, MO, USA), PBK (16110–1-AP-1:100dil; Proteintech, Rosemont, IL, USA), TXNIP (40–3700- 1:100 dil; Thermo Scientific, Waltham, MA, USA), H2AX (10856–1-AP- 1:50 dil; Proteintech), ATF3 (HPA001562–1:50 dil; Sigma-Aldrich Corp.) or a mouse monoclonal antibody against K15 (MA5–11244–1:100 dil; Thermo Scientific). Appropriate secondary antibodies conjugated to Alexa-555 or Alexa-488 (Invitrogen, Carlsbad, CA, USA) at 1:300 dilution were used to detect primary antibodies. DAPI (4′,6-diamidino-2-phenylindole) was used to label nuclei, and mounted using Gelvatol. Images were acquired using a 20X 0.5 EC Plan-Neofluar objective on an epiflourescence microscope system (AxioVision Z1; Carl Zeiss, Thornwood, NY, USA) fitted with an Apotome slide module and a digital camera (AxioCam MRm).
3
Immunohistochemical Quantification of ATF3
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Fresh tissues were fixed, embedded in paraffin, and serially sectioned. Antigen retrieval was conducted by autoclaving the samples at 121 °C for 15 min in the presence of citrate antigen retrieval solution. The samples were incubated with antibodies against ATF3 (1:600; HPA001562, Sigma) overnight at 4 °C followed with immunohistochemical staining kits (Zhongshan Golden Bridge). Control sections were run concurrently with the experimental sections using nonspecific rabbit IgG. Nonspecific staining was not detected in the controls. H-score was calculated with IHC-Profiler52 (link) and ImageJ software (imagej.nih.gov/ij) based on staining intensity and percentage of positive staining cells, and high positive staining scored 3, positive staining scored 2, low positive scored staining 1, and negative staining scored 0. In this study, we only calcite the staining score of the endometrium stromal cells.
4
Zebrafish Embryo Western Blot
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Western blot was performed with standard protocols. Protein was prepared from zebrafish embryos at 9 ss and dissolved in 4× protein SDS–polyacrylamide gel electrophoresis Loading buffer (Takara). Atf3, Perk, and Slc2a1 were detected with anti-ATF3 (HPA001562, Sigma, USA), anti-PERK (C33E10, CST, USA), and anti-Slc2a1 (D3J3A, CST, USA) at a dilution of 1:1000, followed by incubation with an anti-rabbit IgG–horseradish peroxidase antibody (ab97069, Abcam, USA) at a dilution of 1:5000. β-Tubulin was used as the internal control using an anti-β–tubulin antibody (T6199, Sigma, USA), followed by an anti-mouse IgG antibody (62-6520, Invitrogen, USA) diluted 1:1000 and 1:5000 in the block solution, respectively.
5
Immunohistochemical Staining of ATF3
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After the endometrial samples were dewaxed, endogenous peroxidase activity was blocked using freshly prepared PBS containing 0.3% hydrogen peroxide for 15 min. Antigen retrieval was conducted by autoclaving the samples at 121 °C for 15 min in the presence of EDTA (pH = 9.0). The sections were washed with PBS and then incubated with antibodies against ATF3 (1:1000; HPA001562, Sigma) overnight at 4 °C in a humidified chamber. The sections were subsequently rinsed with PBS and incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody at 37 °C for 30 min. HRP activity was detected using diaminobenzidine (Invitrogen), and the sections were counterstained with hematoxylin. Nonspecific rabbit serum (Boster, Wuhan, China) was used as a negative control.
6
Western Blot Analysis of Endometrial Proteins
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Protein extracts were prepared from endometria or cells that were washed with ice-cold phosphate-buffered saline (PBS) and lysed in whole cell lysis buffer (50 mM Tris–HCl [pH 7.6], 150 mM NaCl and 1.0% NP-40) containing a protease inhibitor cocktail and phosphatase inhibitor cocktails 2 and 3 (Sigma). Equal amounts of total protein (30 μg) were separated on 12% (w/v) SDS-polyacrylamide gel and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in Tris-buffered saline solution containing 5% nonfat milk for 1 h and then exposed to the following primary antibodies: ATF3 (1:1000; HPA001562, Sigma, 22 kD), LIF (1:500; L0669, Sigma; 38 kD), STAT3 (1:1000; #12640, Cell Signaling Technology, Danvers, MA, USA, 86 kD), p-STAT3 (1:1000; #9145, Cell Signaling Technology, 90 ~ kD), His-Tag antibody (1:2000; M30111, Abmart, Shanghai, China), and GAPDH (1:10000; AP0063, Bioworld, Nanjing, China; 42 kD). Immunodetection was accomplished using goat anti-rabbit or donkey anti-mouse secondary antibodies and an enhanced chemiluminescence detection kit (Millipore). The density of each band was scanned and measured by ImageJ software with target protein that had been normalized to GAPDH.
7
Immunohistochemical Profiling of ATF3 and TCF3
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TMA tissue sections were deparaffinized, and antigen retrieval was performed in citrate buffer for 3 min at 100 °C. Endogenous peroxidase activity and nonspecific antigens were blocked with peroxidase blocking reagent followed by incubation with primary antibodies against ATF3 (1:300, HPA001562; Sigma–Aldrich, USA) and TCF3 (1:200, sc-133075, Santa Cruz, USA) overnight at 4 °C. After washing, sections were incubated with biotin-labeled secondary antibody and subsequently incubated with a streptavidin-peroxidase complex. The peroxidase reaction was developed using 3,3′-diaminobenzidine (DAB) chromogen solution in DAB buffer substrate (#8059, Cell Signaling, USA). Sections were visualized with DAB, counterstained with hematoxylin, mounted in neutral gum and analyzed using a brightfield microscope. The IHC score was calculated by multiplying the staining intensity grade (grade of 0, 1, 2, or 3 indicating negative, weak-positive, moderate-positive or strong-positive, respectively) by the positive rate score (score of 0, 1, 2, 3, or 4 indicating positive areas of 0–5%, 6–25%, 26–50%, 51–75%, or 76–100%, respectively).
8
Immunofluorescence Analysis of ATF3 in Ishikawa Cells
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Ishikawa cells plated on 18 mm micro-cover glasses were subjected to E2 and MPA for 6 h. Treated or untreated Ishikawa cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After washing with PBS, the cells were permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature. After blocking with 1% bovine serum albumin (BSA) in PBS, the cells were probed for ATF3 and F-actin and then incubated at 4 °C overnight with anti-ATF3 polyclonal antibody (1:1000; HPA001562, Sigma) and F-actin (1:300, P5282, Sigma), with or without Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:200; Invitrogen). Nuclei were stained with 4′,6-diamidine-2-phenylindole (DAPI), which was included in the Vectashield Mounting Medium for Fluorescence with DAPI kit. Images were visualized using a confocal microscope (Leica).
9
Immunoblotting of Protein Extracts
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Proteins were extracted as described previously51 (link). Equal amounts (30 μg) of protein were separated on a 12% SDS-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Immunoblotting was performed by incubating the membranes with primary antibodies against ATF3 (1:1000; HPA001562, Sigma), FOXO1 (1:2000; Cell Signaling Technology, Danvers, MA, United States), His-Tag (1:2000; M30111, Abmart, Shanghai, China), and GAPDH (1:10,000; AP0063, Bioworld Technology, MN, USA).
10
Histological Analysis of Limb Development
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Methods used were as described previously (Tsang et al., 2007 (link)). In brief, limbs were fixed in 4% PFA, followed by demineralization in 0.5M EDTA (pH 8.0) before embedding in paraffin. Slides were stained with Alcian Blue for cartilage matrix and Fast Red for nuclei. Immunofluorescence was performed using antibodies against ATF4 (sc-200, Santa Cruz), ATF3 (HPA001562, Sigma), CHOP (sc-575, Santa Cruz), PPP1R15A (sc-825, Santa Cruz), FGF21 (42189, AIS) and Sox9 (AB5535, Millipore).
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