We resuspended engeletin (>99.0% pure, MW = 434.39) in dimethyl sulfoxide (DMSO). For dose–response studies, rats were randomly assigned to six different treatment groups: sham group, tMCAO group, edaravone group (6 mg/kg) and engeletin groups (15, 30 and 60 mg/kg). Antibodies (HMGB1: ab79823.html">ab79823 , TLR4: ab22048.html">ab22048 , Bcl‐2: ab32124, NF‐κB p65: ab16502, Bax: ab182733 and Cleaved Caspase‐3: ab214430) were acquired via Abcam (Cambridge, MA, United States), and anti‐GAPDH (AF2819) from Beyotime Biotechnology (Shanghai, China).
Ab22048
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab22048 is a protein-specific antibody product. It is designed to detect the target protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab16502, by Abcam (20 mentions)
Ab86299, by Abcam (12 mentions)
Ab32503, by Abcam (7 mentions)
Ab32536, by Abcam (6 mentions)
Ab13847, by Abcam (5 mentions)
Ab9722, by Abcam (4 mentions)
Ab16894, by Abcam (4 mentions)
Ab8227, by Abcam (4 mentions)
Ab133739, by Abcam (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Ab59348, by Abcam (4 mentions)
Ab79823, by Abcam (3 mentions)
Ab2064, by Abcam (3 mentions)
Ab13556, by Abcam (3 mentions)
Ab2068, by Abcam (3 mentions)
Ab32518, by Abcam (3 mentions)
Ab110304, by Abcam (3 mentions)
Ab97051, by Abcam (3 mentions)
Ab233706, by Abcam (3 mentions)
Ab135693, by Abcam (3 mentions)
99 protocols using ab22048
1
Engeletin Neuroprotection in Stroke
2
Immunohistochemical Analysis of Gastric Tissues
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The paraffin-embedded gastric tissues were dewaxed, rinsed with phosphate-buffered saline (PBS, pH 7.4) three times for 5 min and treated with 10% FBS at 37 °C for 30 min. Sequentially, the gastric tissues were dewaxed with dimethyl benzene and an ethanol gradient, washed with phosphate-buffered saline (PBS) for 3 min each time for a total of three times and treated with 10% FBS at 37 °C for 30 min. Next, sections were incubated with primary antibodies against Keap1 (1:500, ab196346, Abcam, Cambridge, UK), Nrf2 (1:200, ab137550, Abcam, Cambridge, UK), TLR4 (1:500, ab22048; Abcam, Cambridge, UK) and NF-κB (1:500, ab16502; Abcam, Cambridge, UK) in a humidified chamber at 4 °C overnight. The sections were incubated with HRP-labelled secondary antibodies for 30 min after being washed with PBS three times. Then, 3,3′-diaminobenzidine (DAB, Sigma-Aldrich, St. Louis, MO, USA) was used as a substrate to visualize positive staining. Finally, images were obtained by using an IX73 Olympus microscope equipped with a DP80 camera (Olympus Corp., Tokyo, Japan).
3
Protein Expression Analysis in Myocardial Infarction
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Rats of each group were sacrificed when the 4-week treatments were finished, and hearts were snap-frozen and stored at −80°C. Myocardial tissues from the infarct border zone (2 mm away from the infarct edge) (Kohno et al., 2008 (link)) were sheared and homogenized in ice-cold lysis buffer system containing RIPA lysate (1 mL, 89900, ThermoFisher, China), protease inhibitors (10 μL, ST506-2, Beyotime, China), and phosphatase inhibitors (10 μL, P1260, Applygen, China). Subsequent steps including protein sample extraction, concentration normalization, and detection of specific protein were conducted according to our previous description (Wu et al., 2017 (link)). The details of primary antibodies are listed below: p-AMPK (Thr172; 40H9, CST, United States), AMPK (D5A2, CST, United States), Cx43 (3512, CST, United States), TGF-β (ab92486, Abcam, China), Collagen-I (14695-1-AP, Proteintech, China), Collagen-III (22734-1-AP, Proteintech, China), IL-1β (ab9722, Abcam, China), IL-6 (ab9324, Abcam, China), TNF-α (ab220210, Abcam, China), TLR-4 (ab22048, Abcam, China), MyD88 (ab2064, Abcam, China), p-P65 (Ser536; ab86299, Abcam, China), and P65 (ab16502, Abcam, China), GAPDH (ab181602, Abcam, China).
4
Immunofluorescence Imaging of Cellular Markers
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NRK-52E cells that had undergone various treatments were fixed in 4% paraformaldehyde and per-mobilized in 0.1% Triton X-100, before they were, respectively, treated with primary mouse monoclonal anti-E-cadherin (E-ca) antibody (Abcam ab1416, 1:100), mouse monoclonal collagen I antibody (Abcam ab260043, 1:100), TLR4 (Abcam ab22048, 1:200), NF-κB (Abcam ab194726, 1:200). After three washes with PBS, the sections were incubated for 2 h with DAR-FITC (1:50) and Texas Red-DAM (1:50) at RT. The fluorescent images were visualized with a Fluoview 300 fluorescence microscope (Olympus, Tokyo, Japan).
5
Immunohistochemical Detection of TLR4, NF-kB, and mTOR
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The main steps of immunohistochemical detection included dewaxing, hydration, 3% hydrogen peroxide treatment, high temperature antigen retrieval, goat serum sealing, primary antibody 4°C overnight, rewarming PBS wash, secondary antibody drop, DAB color development, hematoxylin staining, hydrochloric acid and alcohol differentiation, tap water wash, dehydration, and sealing. Images were captured under a microscope, and the Image-Pro Plus 6.0, Media Cybernetics system was used to detect absorbance and determine integrated option density (IOD)/area of TLR4 (ab22048, Abcam, Cambridge, MA, USA), NF-κB p65 (ab16502, Abcam), or mTOR (ab32028, Abcam), and to perform statistical processing.
6
Immunohistochemical Analysis of Toll-Like Receptors in Endometrial Pathologies
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Serial sections of diameter 3 microns were taken from the paraffin-embedded tissue samples of endometrial cancer, hyperplasia, and benign endometrium. One of these sections was stained with hematoxylin-eosin to confirm the diagnosis. Immunohistochemicals were applied to the tissue samples by an automated IHC/ISH stainer (Leica BOND-MAXTM, New Castle, UK). An anti-TLR2 antibody (anti-TLR2 antibody ab24192, ABCAM, Cambridge, MA, USA), an anti-TLR4 antibody (anti-TLR4 antibody [76B357.1] ab22048, ABCAM, Cambridge, MA, USA), an anti-TLR5 antibody (anti-TLR5 antibody [19D759.2] ab13876, ABCAM, Cambridge, MA, USA), and an anti-TLR6 antibody (anti-TLR6 antibody ab59920, ABCAM Cambridge, MA, USA) were used.
Immunohistochemical evaluation was performed in a semiquantitative manner by the same pathologist. The distribution and intensity of the TLR 2, 4, 5, and 6 staining of the cells were evaluated and compared with the background tumor cells. Staining of the nucleus and/or cytoplasm was considered a positive result. According to the distribution and intensity of the immunohistochemical staining, the samples were subjectively divided into four groups: group 0: no staining, group 1: mild staining, group 2: moderate staining, and group 3: severe staining. The SPSS 16.0 (Chicago, IL) package program was used for statistical analysis, and p<0.05 was considered significant.
Immunohistochemical evaluation was performed in a semiquantitative manner by the same pathologist. The distribution and intensity of the TLR 2, 4, 5, and 6 staining of the cells were evaluated and compared with the background tumor cells. Staining of the nucleus and/or cytoplasm was considered a positive result. According to the distribution and intensity of the immunohistochemical staining, the samples were subjectively divided into four groups: group 0: no staining, group 1: mild staining, group 2: moderate staining, and group 3: severe staining. The SPSS 16.0 (Chicago, IL) package program was used for statistical analysis, and p<0.05 was considered significant.
7
Blocking Phagocytosis Receptors in Sporothrix Interactions
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S. schenckii and S. brasiliensis were analyzed for their interaction with hMDM in the presence of wHS or heat-inactivated human serum (iHS) as well as to evaluate phagocytic index upon blocking CR3, TLRs and Dectin-1. To block CR3, TLR2, TLR4, and Dectin-1 receptors, monoclonal anti-CD11b (Thermo Fisher, MA5-16528), anti-TLR2 (Abcam, ab16894), anti-TLR4 (Abcam, ab22048), and anti-Dectin-1 antibodies (35 (link)) were used, respectively. Briefly, hMDMs were preincubated with fresh DMEM medium supplemented with 10% of wHS for 30 min at 37°C in an atmosphere of 5% of CO2. Monoclonal antibodies were then added in a final concentration of 10 µg/ml and incubated further at 8°C for 1 h. Then, hMDMs were infected with FITC-labeled yeasts of S. schenckii or S. brasiliensis, and the interaction was allowed for a duration of 1 h at 37°C in a CO2 incubator. The hMDMs were washed one time with PBS (GIBCO®, NY, USA) pH 7.4 and incubated for 15 min at ambient temperature with Trypan blue (GIBCO®, NY, USA) at a final concentration of 1.2 mg/ml. The cells were washed twice with PBS and fixed with 1% p-formaldehyde for 15 min at ambient temperature. Finally, hMDMs were washed once with PBS and, with the aid of a cell scraper, were gently released from the wells, suspended in PBS supplemented with 3% fetal bovine serum, and analyzed in a BD FACS-Canto™ II.
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S. schenckii and S. brasiliensis were analyzed for their interaction with hMDM in the presence of wHS or heat-inactivated human serum (iHS) as well as to evaluate phagocytic index upon blocking CR3, TLRs and Dectin-1. To block CR3, TLR2, TLR4, and Dectin-1 receptors, monoclonal anti-CD11b (Thermo Fisher, MA5-16528), anti-TLR2 (Abcam, ab16894), anti-TLR4 (Abcam, ab22048), and anti-Dectin-1 antibodies (35 (link)) were used, respectively. Briefly, hMDMs were preincubated with fresh DMEM medium supplemented with 10% of wHS for 30 min at 37°C in an atmosphere of 5% of CO2. Monoclonal antibodies were then added in a final concentration of 10 µg/ml and incubated further at 8°C for 1 h. Then, hMDMs were infected with FITC-labeled yeasts of S. schenckii or S. brasiliensis, and the interaction was allowed for a duration of 1 h at 37°C in a CO2 incubator. The hMDMs were washed one time with PBS (GIBCO®, NY, USA) pH 7.4 and incubated for 15 min at ambient temperature with Trypan blue (GIBCO®, NY, USA) at a final concentration of 1.2 mg/ml. The cells were washed twice with PBS and fixed with 1% p-formaldehyde for 15 min at ambient temperature. Finally, hMDMs were washed once with PBS and, with the aid of a cell scraper, were gently released from the wells, suspended in PBS supplemented with 3% fetal bovine serum, and analyzed in a BD FACS-Canto™ II.
8
Visualizing Protein Interactions via Immunofluorescence
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For the immunofluorescent staining experiments, the RAW264.7 cells were incubated with 1 μg/mL rhENO1 in TCM for 30 min. The cells were fixed in 4% PFA for 15 min and washed three times with PBS. Samples were blocked with 5% albumin from bovine serum (BSA) with 0.1% Triton X-100 in PBS for 60 min at room temperature and then incubated with primary antibodies overnight at 4 °C, followed by the secondary fluorescently labeled antibodies for one hour at room temperature. The primary antibodies used in this experiment were: anit-ENO1 (ab155102, 1:500) and anti-TLR4 (ab22048, 1:100) from abcam. The secondary antibodies used in this experiment were: Goat anti-Rabbit IgG (H+L) conjugated with Alexa Fluor 594 (R37117, 2 drops diluted in 1 mL PBS) from Thermo Fisher Scientific, and goat anti-mouse IgG (H+L) conjugated with Alexa Fluor 488 (ab150113, 1:200) from abcam. The nuclei were counterstained with DAPI (Invitrogen, Waltham, MA, USA). Images were captured via an inverted fluorescence microscope (Nikon, Japan).
9
TREM1, TLR2, and TLR4 Interaction Analysis
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Cells in the culture plate were first washed with phosphate buffer and were scraped off into 1 mL of cold lysis buffer for IP (Beyotime). After the cell suspension was centrifuged, anti-TREM1 (ab214202; Abcam, Cambridge, MA, USA), anti-TLR2 (ab209216; Abcam, Cambridge, MA, USA), anti-TLR4 (ab22048; Abcam, Cambridge, MA, USA), and control lgG for 1 µg plus Protein A/G beads (Santa Cruz Biotechnology, CA, USA) were, respectively, added to the supernatant for co-immunoprecipitation. The beads were washed with phosphate-buffered saline (PBS) three times, and the immunoprecipitants were assessed via Western blot.
10
Immunofluorescence Analysis of NLRP3 and TLR4
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Paraffin sections of cerebral cortex tissues were prepared, and the expression levels of NLRP3 and TLR4 were detected by an immunofluorescence analysis. After dewaxing and rehydration, the sections were placed in citrate buffer, and the antigens were recovered by a thermally mediated method. The sections were then incubated with 3% BSA for 30 min (Sigma–Aldrich, St. Louis, USA). Then, the sections were incubated overnight at 4 °C with diluted primary antibodies against NLRP3 (MA5–23919, Thermo Fisher Scientific) and TLR4 (ab22048, Abcam, Cambridge, UK). On the second day, the sections were incubated with a FITC-labelled secondary antibody at room temperature for 60 min. DAPI was used to stain the nuclei. Finally, the sections were dehydrated and examined under an inverted fluorescence microscope.
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