Paraffin-embedded lung and kidney tissue sections were deparaffinized, and then immersed in 0.2% Triton X-100 for 45 min as described previously (8 (link)). Following blocking with 10% donkey serum (ab7475, Abcam Inc, Cambridge, MA) in PBS for 1 h, slides were immunostained with rabbit anti-SP-A antibody, anti-TLR-4 (ab13556, Abcam Inc, Cambridge, MA), anti-TNFR1(sc-374186, Santa Cruz Biotechnology, Dallas, Texas). For cell IF analysis, the cells were fixed with 4% paraformaldehyde, and examined with anti-TLR-4(ab13556, Abcam Inc, Cambridge, MA), anti-TNFR1(sc-374186, Santa Cruz Biotechnology, Dallas, Texas) and anti-Megalin antibody (sc-16478, Santa Cruz Biotechnology, Dallas, Texas) to confirm the types of cultured cells and to determine the purity and quantity of proximal tubular epithelial cells. Slides were stained using Alexa 488 (ab150073, Abcam Inc, Cambridge, MA) and/or Alexa 594-conjugated secondary antibodies (A11058, Life Technologies, Eugene, OR) at room temperature for 1 h for fluorescence visualization of primary antibodies.
Ab13556
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab13556 is a laboratory product from Abcam. It is a piece of equipment designed for use in research and scientific applications. No further details about its specific function or intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab16502, by Abcam (14 mentions)
Pvdf membrane, by Merck Group (10 mentions)
Ab32536, by Abcam (9 mentions)
Ab2064, by Abcam (9 mentions)
Ab6721, by Abcam (9 mentions)
Ab205718, by Abcam (9 mentions)
Bca kit, by Thermo Fisher Scientific (9 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Ab8226, by Abcam (7 mentions)
Ripa lysis buffer, by Beyotime (7 mentions)
Ab219413, by Abcam (6 mentions)
Ab86299, by Abcam (6 mentions)
Ab133739, by Abcam (6 mentions)
Ab32503, by Abcam (6 mentions)
Bca protein assay kit, by Beyotime (5 mentions)
Ab8227, by Abcam (5 mentions)
Ab181602, by Abcam (5 mentions)
Ab214185, by Abcam (5 mentions)
Ab213676, by Abcam (4 mentions)
Ab182858, by Abcam (4 mentions)
105 protocols using ab13556
1
Immunofluorescence Staining of Lung and Kidney Tissue
2
Molecular Profiling of Lung Inflammation
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Lung sections were prepared as previously described, then stained with mouse anti-MUC5AC antibody (#ab3649, Abcam), rabbit anti-α-SMA (#ab32575, Abcam), TLR4 (#ab13556, Abcam), rabbit anti-HMGB1 antibody (#ab79823, Abcam), rabbit anti-NLRP3 antibody (#214185, Abcam), rabbit anti-NLRC4 antibody (#A13117, ABclonal) and rabbit anti-Bcl-2 antibody (#A0208, ABclonal). After washing with PBS, slices were incubated with anti-rabbit secondary antibody for 30 minutes at room temperature. Signals were visualized with a DAB peroxidase substrate kit., and the localization of targeted molecules in the lung was assessed under a light microscope. Lung tissue samples were ground to fine powder under liquid nitrogen and lysates were subjected to SDS-PAGE and western blotting for the detection of the following antigens: TLR4 (#ab13556, Abcam), HMGB1 (#ab79823, Abcam), NLRP3 (#214185, Abcam), NLRC4 (#A13117, ABclonal), Bcl-2 (#A0208, ABclonal), procaspase-3 (#A0214, ABclonal), procaspase-8 (#108333, Abcam), cleaved caspase-3 (#9664s, Cell Signaling Technology) and cleaved caspase-8 (#9429, Cell Signaling Technology). After incubation with an IRDye® 680WC-conjugated secondary antibody (LICOR Biosciences), immunoreactive bands were exposed to an Odyssey® CLx Imager for image capture. Data analysis was performed with ImageJ Software.
3
Immunoblotting analysis of TLR4 and NF-κB
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The target cells were used to isolate the proteins. The isolated proteins were deposited onto the PVDF membrane after electrophoresis with SDS-PAGE and then determined by immunoblotting with the specified primary antibodies. TLR4 (1/1000, ab13556, Abcam), NF-κB p65 (1/1000, 8242S, Cell Signaling Technology, Danvers, United States), p-NF-κB p65 (1/500, ab194726, Abcam), and actin (use concentration of 1 μg/ml, ab8226, Abcam, endogenous control) antibodies were used in this study. Antirabbit or antimouse IgG antibodies conjugated to horseradish peroxidase (Abcam) were used to cultivate the membranes. The protein bands were seen using the Thermo Scientific Pierce ECL Western Blotting Substrate.
4
Liver Protein Extraction and Western Blot Analysis
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The liver was weighed and ground with liquid nitrogen. The milled tissue powder was added into lysis solution containing RIPA (tissue weight: volume of lysis solution 20 mg:150–250 μL) for lysis. BCA protein detection kit was used to detect protein concentration. The total protein (10 μg) of each sample was added to SDS-PAGE gel electrophoresis to separate the protein and transferred to PVDF membrane (Millipore). The PVDF membrane containing protein was incubated in a closed solution for 2 hours and then incubated in the required primary antibody, including TLR4 antibody (1 : 500, ab13556; Abcam), MyD88 antibody (1 : 1000, ab219413; Abcam), TRAF6 (1 : 1000, ab33915; Abcam), IκB antibody (1 : 2000, ab76429; Abcam), P-IκB antibody (1 : 1000, 2859 S; Abcam), NF-κB antibody (1 : 2000, ab16502; Abcam), and β-actin antibody (1 : 10000, 20536-1-AP; Proteintech), incubated at 4°C overnight. After incubating with HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (1 : 5000, SA00001-2, Proteintech) and HRP-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (1 : 5000, SA00002-1, Proteintech) for 1 h, the PVDF membrane was washed with solution TBST, treated with chemiluminescence reagent, taken photos by exposure, and performed quantitative analysis with Image-Pro Plus 6.0.
5
Quantifying TLR-4 Expression in Cells
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Maxisorb Immunoplates were coated with 0.5 μg/ml of mouse monoclonal to TLR-4 (Abcam - ab22048) (epitope 100 to 200) and blocked with 5% milk powder. Samples were applied (one hour at room temperature) and the amounts of TLR-4 were measured using a rabbit polyclonal IgG to TLR-4 (Abcam - ab13556) (epitope 420 to 435), followed by anti-rabbit IgG conjugated to alkaline phosphatase and 1 mg/ml 4-nitrophenyl phosphate. Absorbance was measured on a microplate reader at 405 nm. Results were reported as% TLR-4 where 100% = amount of TLR-4 in cell extracts from control RAW 264 cells.
6
YfeA modulates signaling pathways in RAW 264.7 macrophages
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RAW 264.7 macrophages were treated with or without YfeA (20 µg/mL) for 12 h, the total cellular protein was extracted using a Protein Extracting Kit (Solarbio, Beijing, China). Aliquots corresponding to 50 µg of each sample were analyzed using Western blotting (WB). To analyze the YfeA-induced signal transduction residue phosphorylation, the primary monoclonal antibodies, including Erk1/2 (rabbit, 42/44 kDa; 1:2000; Abcam ab184699), p-Erk1/2 (rabbit, p-ERK1: Thr202/Tyr204, p-ERK2: Thr185/Tyr187; 42/44 kDa; 1:2000; Abcam ab278538), p38-MAPK (rabbit, 41 kDa; 1:1000; Abcam ab31828), p-p38-MAPK(rabbit, Tyr182; 41 kDa; 1:500; Abcam ab47363), JNK (rabbit, 48 kDa; 1:1000; Abcam ab179461), p-JNK (rabbit, Tyr185/223; 48 kDa; 1:10,000 Abcam ab76572), p65 (NF-κB; rabbit, 60 kDa; 1:2000; Abcam ab16502), p-p65 (NF-κB; rabbit, Ser536; 60 kDa; 1:5000; Abcam ab86299), TLR2 (rabbit, 89 kDa; 1:500; Abcam ab213676), and TLR4 (rabbit, 89 kDa; 1:500; Abcam ab13556) were applied at this stage.
7
Immunohistochemical Analysis of Kidney Tissue
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Formalin-fixed, dehydrated, paraffin-embedded kidney sections (4 μm) were deparaffinized, rehydrated, and antigen-retrieved. The slides were then blocked by 2.5% normal goat serum and incubated with primary antibodies anti-FABP4 (1:200, 12802-1-AP, Proteintech Group, Chicago, USA), anti-TLR4 (1:200, Ab13556, Abcam, MA, USA), and anti-phospho-c-Jun (1:200, ET1608-4, HuaAn Biotechnology, Hangzhou, China) at 4°C. The slides were washed thrice in PBS and stained using VECTASTAIN ABC Kit (Vector, Burlingame, CA, USA). Images were captured using an AxioCamHRc digital camera (Carl Zeiss, Jena, Germany) at ×200 and ×400 magnifications with ZEN 2012 microscopy software (blue edition). The positive area of immunohistochemistry staining was calculated at ×200 magnification with ImageJ software (version 1.51, Wayne Rasband, National Institutes of Health, USA).
8
Quantitative Western Blot Analysis
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Western blot was performed as previously described31 (link). Briefly, cerebral cortex samples were collected, homogenised and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis on 10% polyacrylamide gels. A BCA Protein Assay Kit (Beyotime) was used to measure protein concentrations using the bicinchoninic acid method. After separation, protein samples were transferred onto immobilon nitrocellulose membranes. The membranes were blocked with 5% nonfat milk at room temperature for 1 h and then incubated with the following primary antibodies overnight at 4 °C: rabbit anti-β-actin (1:1000, rabbit polyclonal, Abcam, ab8227), rabbit anti-caspase-3 (1:2000, rabbit polyclonal, Abcam, ab184787), rabbit anti-Bax (1:2.000, ab182733), rabbit anti-Bcl2 (1:2.000, ab182858), rabbit anti-TLR4 (1:1000, rabbit polyclonal, Abcam, ab13556) and rabbit anti-NF-κB p65 (1:1000, rabbit monoclonal, Abcam, ab32536). After washing the membranes with TBST three times, horseradish peroxidase-conjugated goat antirabbit immunoglobulin G (IgG) or goat antimouse IgG secondary antibodies (1:5000) were used, and the membranes were incubated using secondary antibodies at room temperature for 1.5 h. The protein bands were detected using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA) and quantified with ImageJ software.
9
Western Blot Analysis of TLR4 and CD11b
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After 24 h of treatment,
adherent
cells were washed with ice-cold PBS and homogenized in lysis buffer.
The protein concentration in the supernatant was determined using
a BCA protein assay kit (Thermo Fisher Scientific), and proteins were
resolved on 10% SDS/PAGE gels. After transfer to 0.45 μm PVDF
membranes, the membranes were blocked for 1 h in blocking buffer and
then incubated overnight at 4 °C with the primary TLR4 or CD11b
antibody (TLR4: ab13556, Abcam; CD11b: ab133357, Abcam; MyD88: ab2064,
Abcam; Actin: sc-1616, Santa Cruz Biotechnology) in the same buffer.
The blots were then washed in TBS-T (TBS buffer containing 0.1% Tween-20),
incubated for 1 h with fluorescently labeled secondary antibodies,
and detected using Odyssey Imaging Systems.
adherent
cells were washed with ice-cold PBS and homogenized in lysis buffer.
The protein concentration in the supernatant was determined using
a BCA protein assay kit (Thermo Fisher Scientific), and proteins were
resolved on 10% SDS/PAGE gels. After transfer to 0.45 μm PVDF
membranes, the membranes were blocked for 1 h in blocking buffer and
then incubated overnight at 4 °C with the primary TLR4 or CD11b
antibody (TLR4: ab13556, Abcam; CD11b: ab133357, Abcam; MyD88: ab2064,
Abcam; Actin: sc-1616, Santa Cruz Biotechnology) in the same buffer.
The blots were then washed in TBS-T (TBS buffer containing 0.1% Tween-20),
incubated for 1 h with fluorescently labeled secondary antibodies,
and detected using Odyssey Imaging Systems.
10
Quantifying microglial TLR4 and CD11b expression
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After treatment with LPNP-TLR4
siRNA or LPNP-CD11b siRNA at different concentrations in the Lab-Tek
chamber (155411PK734-2062 vwr), the microglial cells were washed with
PBS and fixed with 1% paraformaldehyde solution for 10 min. After
rinsing the cells with TBS, the TLR4 (Abcam, ab13556, 1:200) or CD11b
(Abcam, ab133357, 1:200) primary antibody was incubated (in SUMI buffer
(2.5 mg/mL gelatin, 0.5% Triton, dissolved in TBS)) with the cells
overnight at 4 °C. After rinsing with TBS, the secondary antibody
(biotinylated anti-rabbit, Vector Lab, BA1100, 1:400) was incubated
with the cells for 1 h at room temperature. The cells were washed
with TBS and incubated with streptavidin Alexa Fluorophore (for TLR4:
streptavidin Alexa Fluorophore 594, Jackson ImmunoResearch, 016-580-084,
1:300; for CD11b: streptavidin Alexa Fluorophore 488, Invitrogen,
s32354, 1:300) for 1 h at room temperature. After washing with TBS,
the cells were incubated with DAPI (Sigma-Aldrich; 1:1000) for 10
min. Following washes in TBS, the fluorescence signals in cells were
detected using a Leica TCS SP5 inverted confocal microscope.
siRNA or LPNP-CD11b siRNA at different concentrations in the Lab-Tek
chamber (155411PK734-2062 vwr), the microglial cells were washed with
PBS and fixed with 1% paraformaldehyde solution for 10 min. After
rinsing the cells with TBS, the TLR4 (Abcam, ab13556, 1:200) or CD11b
(Abcam, ab133357, 1:200) primary antibody was incubated (in SUMI buffer
(2.5 mg/mL gelatin, 0.5% Triton, dissolved in TBS)) with the cells
overnight at 4 °C. After rinsing with TBS, the secondary antibody
(biotinylated anti-rabbit, Vector Lab, BA1100, 1:400) was incubated
with the cells for 1 h at room temperature. The cells were washed
with TBS and incubated with streptavidin Alexa Fluorophore (for TLR4:
streptavidin Alexa Fluorophore 594, Jackson ImmunoResearch, 016-580-084,
1:300; for CD11b: streptavidin Alexa Fluorophore 488, Invitrogen,
s32354, 1:300) for 1 h at room temperature. After washing with TBS,
the cells were incubated with DAPI (Sigma-Aldrich; 1:1000) for 10
min. Following washes in TBS, the fluorescence signals in cells were
detected using a Leica TCS SP5 inverted confocal microscope.
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