Total protein was collected according to the methods described in our previous report. The protein concentration was assessed using a Thermo Fisher bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were separated using 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with primary antibodies diluted to the recommended concentration, including mouse anti-α-SMA (Abcam, Ab240654, 1:500), rabbit anti-collagen I (Abcam, Ab260043 1:500), rabbit anti-SENP1 (Abcam, Ab236094, 1:500), rabbit anti-β-Catenin (Abcam, Ab32572, 1:500), and rabbit anti-GLI1 (Abcam, Ab217326, 1:500), rabbit anti-SPC (Abcam, Ab211326, 1:500) overnight at 4 °C. The membranes samples were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1.5 h, and the protein bands were detected using a chemiluminescence device.
Ab217326
Manufactured by Abcam
Sourced in United States
Ab217326 is a rabbit polyclonal antibody. The core function of this product is to detect the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Ab53281, by Abcam (3 mentions)
Ab53715, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab236465, by Abcam (2 mentions)
Ab260043, by Abcam (1 mentions)
Ab240654, by Abcam (1 mentions)
Ab236094, by Abcam (1 mentions)
Ab211326, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Ab32053, by Abcam (1 mentions)
Af1009, by Abcam (1 mentions)
Ab51254, by Abcam (1 mentions)
Ab68155, by Abcam (1 mentions)
Ab58632, by Abcam (1 mentions)
Haematoxylin, by Beyotime (1 mentions)
Neutral gum, by Beyotime (1 mentions)
Ab75150, by Abcam (1 mentions)
Ab135240, by Abcam (1 mentions)
Ab92552, by Abcam (1 mentions)
Ab184787, by Abcam (1 mentions)
9 protocols using ab217326
1
Comprehensive Protein Expression Analysis
2
Immunohistochemical Analysis of Hedgehog Pathway
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The collected samples were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemical staining was performed using 4 μm thick wart and foreskin sections. First, the sections were placed on a microscope slide, baked for about 30 min, deparaffinized in xylene and ethanol, and subsequently placed into the immunohisto stainer (Leica bond‐max). The sections were then incubated with 3% H2O2 solution for 20 min to inhibit endogenous peroxidase. Thereafter, the sections were treated with 3% goat serum for 30 min and then incubated with the primary antibodies, namely rabbit anti‐SHh monoclonal antibody (Abcam, ab53281, diluted at 1:500), anti‐Ptch1 polyclonal antibody (Abcam, ab53715, diluted 1:100), a polyclonal antibody of Gli1 (Abcam, ab217326, diluted 1:800), and anti‐Cyclin B1 monoclonal antibody (Abcam, ab32053, diluted 1:250). After removing the primary antibodies, they were incubated with the secondary antibody (DAKO, antGene ANT058) for 25 min. The sections without primary antibodies were used as the negative control. Sections were stained in diaminobenzidine solution, counterstained in hematoxylin solution, blued in bluing reagent, dehydrated in a series of ethanol and xylene, and resin sealed. The slides were visualized using a light microscope. The average optical density (AOD) of the photos was calculated by the Image J software.
3
Immunohistochemical Analysis of HIF-1α and GLI1
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The paraffin-embedded fresh tissue specimen was stained by immunohistochemical staining according to the standard procedure. Soak the sample in 3% hydrogen peroxide solution at room temperature and incubate with the primary antibody overnight at 4°C. The secondary antibody labeled with biotin was incubated at room temperature for 2 hours and washed with PBS 3 times, incubated with diaminobenzidine (DAB) and hematoxylin, and observed and photographed under the microscope. The primary antibody used included the following: anti-HIF1α (Affinity, AF1009, 1 : 100) and anti-GLI1 (Abcam, ab217326, 1 : 100). The immunostaining score of HIF-1α and GLI1 protein in endometrial tissues is presented in Supplemental Table 4 .
4
Immunohistochemical Analysis of Mouse Lumbar Spine
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The mouse lumbar spines (L3–L6) were used for IHC staining. After deparaffinization and hydration, the slices were placed in the citrate buffer and heated to 95°C for 10 min; 3% H2O2 was then used to inactivate peroxidases. After blocking nonspecific antigens with 10% goat serum, we incubated the slices with primary antibody dilution solution at 4°C overnight. The primary antibodies used were as follows: PTH1R (1 : 100, ab75150, Abcam, USA), collagen II (1 : 300, ab34712, Abcam, USA), collagen X (1 : 100, ab58632, Abcam, USA), superoxide dismutase 1 (SOD1, 1 : 1000, ab51254, Abcam, USA), SOD2 (1 : 200, ab68155, Abcam, USA), proliferating cell nuclear antigen (PCNA, 1 : 300, ab92552, Abcam, USA), Ki67 (1 : 500, ab15580, Abcam, USA), caspase3 (1 : 1000, ab184787, Abcam, USA), caspase9 (1 : 300, ab202068, Abcam, USA), SHH (1 : 500, ab135240, Abcam, USA), and GLI1 (1 : 200, ab217326, Abcam, USA). The slices were then washed 3 times with phosphate buffer solution (PBS) and incubated with secondary antibody dilution (Goat Anti-Rabbit IgG, 1 : 500, 12-348, Sigma Aldrich, USA; Goat Anti-Mouse IgG, 1 : 500, 12-349, Sigma Aldrich, USA) at room temperature for 1 h. Diaminobenzidine was used to develop the colour. Finally, the cell nuclei were stained with haematoxylin (Beyotime, China), and the slices were mounted with neutral gum (Beyotime, China) after dehydration.
5
Immunohistochemical evaluation of Hedgehog pathway
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Paraffin tissue sections were preheated in an incubator at 65°C for 5 h, dewaxed
with conventional xylene, repaired in a water bath inside a pressure cooker, and
blocked with a peroxidase blocker for 30 min.9 (link) The samples were then
incubated overnight at 4°C with the primary antibodies rabbit anti-human Shh
(ab53281, Abcam), rabbit anti-human Gli1 (ab217326, Abcam), and rabbit
anti-human Cyr61 (ab228592, Abcam). The slides were then incubated with
horseradish peroxidase conjugated goat anti-rabbit IgG secondary antibody for
30 min at 37°C. Finally, the diaminobenzidine (DAB) color reaction was observed.
Two senior pathologists evaluated the immunohistochemical staining results. The
staining intensity was scored as follows: 0 point for negative, 1 point for weak
positive, 2 points for positive, and 3 points for strong positive. The staining
intensity score was multiplied by the percentage of positive cells to obtain the
final H-Score, and the median H-Score served as the positive-negative
cut-off.10 (link) Overall survival (OS) was calculated from the surgical
treatment date to the end of follow-up or date of death.
with conventional xylene, repaired in a water bath inside a pressure cooker, and
blocked with a peroxidase blocker for 30 min.9 (link) The samples were then
incubated overnight at 4°C with the primary antibodies rabbit anti-human Shh
(ab53281, Abcam), rabbit anti-human Gli1 (ab217326, Abcam), and rabbit
anti-human Cyr61 (ab228592, Abcam). The slides were then incubated with
horseradish peroxidase conjugated goat anti-rabbit IgG secondary antibody for
30 min at 37°C. Finally, the diaminobenzidine (DAB) color reaction was observed.
Two senior pathologists evaluated the immunohistochemical staining results. The
staining intensity was scored as follows: 0 point for negative, 1 point for weak
positive, 2 points for positive, and 3 points for strong positive. The staining
intensity score was multiplied by the percentage of positive cells to obtain the
final H-Score, and the median H-Score served as the positive-negative
cut-off.10 (link) Overall survival (OS) was calculated from the surgical
treatment date to the end of follow-up or date of death.
6
Western Blot Analysis of Hedgehog Signaling
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The cells were collected through centrifugation and resuspended with RIPA (50 mmol/l Tris-HCl, pH 7.5, 150 mmol/l NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS). They were ultrasonicated at 12,000 r/min and centrifuged at 4°C for 10 min. Total protein concentration was determined in accordance with the instructions of the BCA kit. SDS-PAGE was performed with 30 µg of each sample. Protein was transferred to a PVDF membrane and blocked with 5% skimmed milk at room temperature for 1 h. The samples were incubated with the primary antibodies of Gli1 (ab217326, 1:1,000 dilution; Abcam), Gli2 (ab277800, 1:1,000, Abcam), and Rab23 (ab230200, 1:1,000, Abcam) separately. GAPDH was used as an internal reference. The samples were kept at 4°C overnight. The membranes were washed with TBST and incubated with secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. Relative protein expression after ECL development was analyzed by using QuantityOne software (v4.6.7) (Bio-Rad Laboratories, Inc.) after internal reference correction.
7
Western Blot Analysis of Hedgehog and EMT Markers
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Total proteins were extracted from the tissues and cells using RIPA lysis buffer (Beyotime, Shanghai, China) and measured the protein concentration with BCA Protein Assay Kit (Beyotime). The protein samples were then separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated overnight at 4°C with the following primary antibodies (all from Abcam, Cambridge, MA, USA): anti-PTCH (1:1000, ab53715), anti-GLI1 (1:1000, ab217326), anti-SMO (1:1000, ab236465), anti-E-cadherin (1:1000, ab231303), anti-N-cadherin (1:1000, ab76011), anti-vimentin (1:1000, ab92547), anti-β-catenin (1:1000, ab32572), anti-BAX (1:1000, ab32503), anti-BCL2 (1:1000, ab32124), anti-Cleaved caspase 3 (1:500, ab2302) and anti-GAPDH (1:5000, ab8245). After washing with Tris-buffered saline with Tween (Beyotime), the membranes were incubated with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:20,000, ab6721; Abcam) for 1 h at room temperature. Protein bands were visualized using an electrochemiluminescence detection system (Tanon, Shanghai, China) and analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA).
8
Protein Expression Analysis using Western Blot
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Treated cells were collected and lysed by using RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA). After centrifugation for protein extraction, proteins were separated in 10% SDS-PAGE gels, and then these proteins were further transferred to PVDF membrane (Millipore, Burlington, MA, USA). Then the membranes were incubated overnight at 4° C with primary antibodies of anti-Shh (1:1000, ab53281, Abcam, Cambridge, UK), anti-PARP (1:1000, ab191217, Abcam, Cambridge, UK), anti-Cleaved PARP (1:1000, ab225715, Abcam, Cambridge, UK), anti-Bcl-2(1:1000, ab182858, Abcam, Cambridge, UK), anti-GAPDH (1:1000, ab9485, Abcam, Cambridge, UK), anti-MMP-2 (1:1000, ab92536, Abcam, Cambridge, UK), anti-MMP-9 (1:1000, ab283575, Abcam, Cambridge, UK), anti-Smoothened (1:1000, ab236465, Abcam, Cambridge, UK), anti-Gli1 (1:1000, ab217326, Abcam, Cambridge, UK), anti-Patched / PTCH1 (1:1000, ab53715, Abcam, Cambridge, UK), anti-N-myc (1:1000, ab189528, Abcam, Cambridge, UK), anti-Ki67 (1:1000, ab16667, Abcam, Cambridge, UK), anti-Cyclin D1 (1:1000, ab16663, Abcam, Cambridge, UK), or anti-PCNA (1:1000, ab29, Abcam, Cambridge, UK). After incubation with HRP-conjugated secondary antibodies, the bands were visualized by Chemiluminescent substrate ECL (Genbio, Suzhou, China).
9
Protein Expression Analysis by Western Blot
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Western blotting was performed according to standard protocols. Equivalent amounts of protein were separated by electrophoresis on a 4% to 15% resolving gel (Mini-PROTEAN TGX Stain-Free Protein Gels; Bio-Rad) and transferred to nitrocellulose membranes. Membranes were incubated overnight with anti-Zyxin (EPR4302, rabbit, ab109316; dilution 1:2 000; Abcam), anti-CD99 (12E7; mouse, sc-53148; dilution 1:2 000; Santa Cruz Biotechnology), anti-GLI1 (rabbit, ab217326; dilution 1:1,000; Abcam), anti-GAPDH (FL-335, rabbit, catalog No. sc-25778, RRID: AB_10167668, dilution 1:10,000; Santa Cruz Biotechnology), and anti-Lamin B (C-20, goat, catalog No. sc-6216, RRID: AB_648156, dilution 1:10,000; Santa Cruz Biotechnology) primary antibodies. Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit (NA9340V), sheep anti-mouse (NA9310V) both purchased from antibodies (GE Healthcare) or anti-goat (catalog No. sc-2020, RRID: AB_631728sc-2020; Santa Cruz Biotechnology) secondary antibodies were used (diluition 1:10,000). Proteins were visualized with an enhanced chemiluminescence (ECL) Western Blotting Detection System (Euroclone).
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