The LUAD-tissue samples were obtained from Shanghai Zhuoli Biotechnology Co., Ltd (Zhuoli Biotechnology Co, Shanghai, China). The IHC staining in this study was followed by previous report [31] (link). Briefly, after deparaffinizing and hydrating, the sections with four micrometers thick were incubated with the anti-TXNIP (1:200,proteintech,18243-1-AP), anti-GPX4(1:200,ABclonal,A1933), anti-NCOA4(1:200,ABclonal,A5695) primary antibodies at 4 °C overnight followed by incubation with secondary antibodies and diaminobenzidine(DAB) (Maixin,DAB-0031) staining. The expressions of positive cells were analyzed by ImageJ software and the percentage of positive cells (including high positive and positive cells) was scored as 0 (no positive cells), 1 (≤10 % of positive cells), 2 (11 %∼50 % of positive cells), or 3 (>50 % of positive cells). The staining intensity was scored as 0 (negative), 1+ (weakly positive), 2+ (moderately positive), or 3+ (strongly positive). The final expression scores of TXNIP,GPX4 and NCOA4 were evaluated by combining the staining intensity and the percentage of positive cells. High expression in tumor cells was defined as total scored≥2 and low expression was defined as total score<2.
Dab 0031
Manufactured by Maixin Group
Sourced in China
The DAB-0031 is a piece of laboratory equipment. It is designed for performing specific tasks in a laboratory setting. The core function of this product is to facilitate certain experimental or analytical procedures. No further details about its intended use or capabilities are provided.
Automatically generated - may contain errors
Lab products found in correlation
Dig 3009, by Maixin Group (2 mentions)
Mab1281, by Merck Group (1 mentions)
Ab61682, by Abcam (1 mentions)
Dab kit, by Maixin Group (1 mentions)
Sp 9001, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Rm2265 microtome, by Leica (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Bx50 microscope, by Olympus (1 mentions)
Sp kit b1, by Maixin Group (1 mentions)
Mbs9406181, by MyBioSource (1 mentions)
Ab7090, by Abcam (1 mentions)
Gb11132, by Wuhan Servicebio Technology (1 mentions)
Gb11117, by Wuhan Servicebio Technology (1 mentions)
Gb11247, by Wuhan Servicebio Technology (1 mentions)
Ab6671, by Abcam (1 mentions)
Epr3507, by Abcam (1 mentions)
Gb11519, by Wuhan Servicebio Technology (1 mentions)
Kit 9707, by Maixin Group (1 mentions)
Ab9722, by Abcam (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
10 protocols using dab 0031
1
Immunohistochemical Evaluation of TXNIP, GPX4, and NCOA4 in LUAD
2
Immunohistochemistry of Paraffin Sections
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Paraffin-embedded samples were cut into sections of 5μm. Antigen retrieval was achieved by immersing them in 0.1mol/L citrate (pH 6.0) and incubating in an 800-W microwave oven for 15 minutes. MaxvisionTM IHC kit and DAB kit (KIT-5001 and DAB-0031, Maixin Biotech., China) were used. In brief, the sections were blocked for 30 minutes in 5% serum and incubated by the first antibodies i.e. goat anti-human Perilipin A (Ab61682, Abcam), mouse anti-human GFP (66002-1-Ig, CMC) and mouse anti-human nuclear antigen (MAB1281, Millipore) antibodies, then incubated by the second antibodies. While in the blank control group, PBS was used to replace the first antibodies.
3
Immunohistochemical Staining of PGC and MG7 Antibodies
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PGC antibody (anti-pepsinogen C antibody, product name 2D5) was kindly provided by the Institute of Clinical Examination of Japan. MG7 monoclonal antibody was kindly provided by the Digestive Disease Research Laboratory of the Fourth Military Medical University (Xijing Hospital of Digestive Diseases). Immunohistochemical staining was performed using the two-step streptavidin-peroxidase method (Maixin, Kit-9801D2, Fuzhou, China).
Four-μm-thick tissue sections from paraffin-embedded tissues were mounted onto poly-L-lysine-coated glass slides and then baked at 70 °C overnight. The tissue sections were deparaffinized in xylene, rehydrated in ethanol, and then immersed in citrate buffer for antigen retrieval. Endogenous peroxidase was quenched using 3% hydrogen peroxide for 10 min. To decrease nonspecific staining, 2% normal non-immune animal serum was subsequently used to block tissue collagen for 10 min. The tissue sections were then incubated at 4 °C overnight with either the PGC or MG7 antibodies (5 μg/mL) or phosphate buffer solution (PBS) alone. Next, the sections were incubated with biotinylated anti-mouse secondary antibody for 30 min and then washed with PBS. The slides were stained with diaminobenzidine chromogenic reagent (DAB-0031, Maixin Inc.) for 3−10 min, and the reaction was stopped with water.
Four-μm-thick tissue sections from paraffin-embedded tissues were mounted onto poly-L-lysine-coated glass slides and then baked at 70 °C overnight. The tissue sections were deparaffinized in xylene, rehydrated in ethanol, and then immersed in citrate buffer for antigen retrieval. Endogenous peroxidase was quenched using 3% hydrogen peroxide for 10 min. To decrease nonspecific staining, 2% normal non-immune animal serum was subsequently used to block tissue collagen for 10 min. The tissue sections were then incubated at 4 °C overnight with either the PGC or MG7 antibodies (5 μg/mL) or phosphate buffer solution (PBS) alone. Next, the sections were incubated with biotinylated anti-mouse secondary antibody for 30 min and then washed with PBS. The slides were stained with diaminobenzidine chromogenic reagent (DAB-0031, Maixin Inc.) for 3−10 min, and the reaction was stopped with water.
4
Histological and Immunohistochemical Analysis of Ligamentum Flavum and Synovium
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LBs and synovium specimens were fixed in 4% paraformaldehyde solution. For decalcification, LBs were immersed in a solution containing 10% ethylene diamine tetraacetic acid (EDTA) for 3 months. After a series of classic treatments for histological observations, the paraffin-embedded sections of 4 μm-thick were acquired, and disposed with haematoxylin and eosin (HE) staining.
Streptavidin-peroxidase conjugated method was applied for IHC observations, as described previously29 (link). Antigen was retrieved using pepsin (DIG-3009, Maixin, China) at 37°C for 30 min. Rabbit-originated antibodies against CD34 (1:400, ZA-0550, Zhongshan Golden Bridge Biotechnology Co., Ltd., China), human TGF-β3 (1:200, 18942-1-AP, Proteintech), and human FGF-2 (1:500, ZS-79, Zhongshan Golden Bridge Biotechnology Co., Ltd., China) were used as primary antibodies and incubated at 4°C for 18 h. The histological sections were then stained by the anti-rabbit streptavidin-peroxidase kit (SP-9001, Zhongshan Golden Bridge Biotechnology Co., Ltd., China). Finally, color development was achieved by reacting with 3, 3′-diaminobenzidine (DAB, 0031, Maixin, China). Hematoxylin was used for counterstaining.
Streptavidin-peroxidase conjugated method was applied for IHC observations, as described previously29 (link). Antigen was retrieved using pepsin (DIG-3009, Maixin, China) at 37°C for 30 min. Rabbit-originated antibodies against CD34 (1:400, ZA-0550, Zhongshan Golden Bridge Biotechnology Co., Ltd., China), human TGF-β3 (1:200, 18942-1-AP, Proteintech), and human FGF-2 (1:500, ZS-79, Zhongshan Golden Bridge Biotechnology Co., Ltd., China) were used as primary antibodies and incubated at 4°C for 18 h. The histological sections were then stained by the anti-rabbit streptavidin-peroxidase kit (SP-9001, Zhongshan Golden Bridge Biotechnology Co., Ltd., China). Finally, color development was achieved by reacting with 3, 3′-diaminobenzidine (DAB, 0031, Maixin, China). Hematoxylin was used for counterstaining.
5
HIF-1α Expression in Disc Tissues
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Disc specimens were fixed in 4% of paraformaldehyde for 24 hrs and then embedded in paraffin. Sagittal sections (4 μm) were cut with a LeicaRM2265 microtome (Leica, Wetzlar, Germany). Then, the sections were treated with haematoxylin and eosin (HE) staining. Immunohistochemical staining was performed with mouse anti‐human HIF‐1α. The sections were incubated with pepsin (DIG‐3009; Maixin, Fuzhou, China) for 30 min. at 37°C, and then incubated with 3% of H2O2 for 30 min. Non‐specific binding was blocked with a goat blocking serum. The sections were incubated with antibody at 4°C overnight in a humidified chamber. The sections were then washed with PBS and stained by antimouse streptavidin–peroxidase kit (SP‐9001, Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). At last, the sections were coloured by reacting with 3,3‐diaminobenzidine (DAB‐0031, Maixin). Haematoxylin was used for counter‐staining for light microscopy.
6
Antler Development and Immunohistochemistry
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Three-year-old sika deer (Cervus Nippon) (n = 3) were used in this study and one antler was collected from each animal (ethics number: CAAS201415). The antler was harvested using traditional Chinese antler harvesting procedures at around 35 days of growth, which is approximately half way through the antler growth stage. The distal 5-cm tip of the main beam of each antler was collected and sectioned sagittally along the median plane [20 (link)]. Samples were then fixed in 4% paraformaldehyde (Sigma, China, 158,127-500G) and processed for histology as previously described [21 (link)]. Immunohistochemistry was performed on the sagittal sections of paraffin-embedded antler tips. After being deparaffinized with xylene, endogenous peroxidase activity was quenched with 0.3% H2O2, and antigen retrieval was performed with 0.01 M citric acid at 95 °C for 20 min. Sections were then incubated with anti-TMSB10 antibody or anti-rabbit IgG (isotype control) overnight at 4 °C (antibody information is shown in Table 1 ). After warming and rinsing, sections were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min at 37 °C. Finally, sections were stained with diaminobenzidine (Maixin, China, DAB-0031) and counterstained with hematoxylin.
7
Immunohistochemical Analysis of Tumor Markers
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Nude mice tumor specimens were fixed with 10% neutral formalin and embedded in paraffin, and 4-μm-thick sections were prepared. Immunostaining was performed using the avidin–biotin–peroxidase complex method (Ultrasensitive™, MaiXin, Fuzhou, China). Paraffin sections were dewaxed in xylene and rehydrated in graded alcohols. Antigen retrieval was performed by heating the sections for 1.5 min in 0.01 mol/L citrate buffer, pH 6.0. Non-specific staining was reduced by incubation in blocking buffer containing goat serum (SP KIT-B1; Maixin-Bio, Fuzhou, China) for 30 min. Then, the sections were incubated with α-Fetoprotein, Smooth muscle, βIII tubulin antibody overnight at 4°C. The following day, the sections were incubated with appropriate secondary antibodies for 30 min. The reaction was visualized using DAB (DAB-0031; Maixin-Bio) plus chromogen. Specimens were examined using a BX50 microscope (Olympus). For serum controls, 1% BSA in PBS was used in place of the primary antibody as a negative control.
8
Spatial Distribution of ZNF33B in Ovaries
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The ovaries were dissected and fixed in 4 % neutral formalin, dehydrated,
and embedded in paraffin, and then serial sections were made.
Immunohistochemistry analysis was conducted on the sectioned ovaries to
determine the spatial distribution of ZNF33B. The sliced samples were boiled
in citric acid sodium citrate buffer (0.02 M; pH 6) in an induction cooker
for 10 min to recover antigens. Then, the samples were cooled to room
temperature. The samples were incubated with 3 % hydrogen peroxide for 30 min
to block endogenous peroxidases and then incubated with Rabbit ZNF33B
polyclonal antibody (MyBioSource, MBS9406181, ). The slides were washed
three times with phosphate-buffered saline (PBS) and incubated
with pre-adsorbed HRP (Abcam, AB7090). The immunohistochemistry reaction could be
identified using the DAB chromogenic reagent kit (Maixin Biotech, DAB-0031)
and counterstained with hematoxylin staining solution. Normal non-immune
serum was used as the negative control.
and embedded in paraffin, and then serial sections were made.
Immunohistochemistry analysis was conducted on the sectioned ovaries to
determine the spatial distribution of ZNF33B. The sliced samples were boiled
in citric acid sodium citrate buffer (0.02 M; pH 6) in an induction cooker
for 10 min to recover antigens. Then, the samples were cooled to room
temperature. The samples were incubated with 3 % hydrogen peroxide for 30 min
to block endogenous peroxidases and then incubated with Rabbit ZNF33B
polyclonal antibody (MyBioSource, MBS9406181, ). The slides were washed
three times with phosphate-buffered saline (PBS) and incubated
with pre-adsorbed HRP (Abcam, AB7090). The immunohistochemistry reaction could be
identified using the DAB chromogenic reagent kit (Maixin Biotech, DAB-0031)
and counterstained with hematoxylin staining solution. Normal non-immune
serum was used as the negative control.
9
Immunohistochemical Profiling of Inflammation Markers
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The sections were microwave antigen‐retrieved in citrate solution. For immunohistochemistry (IHC), commercial IHC kits (KIT‐9707; Maixin) were used according to the manufacturer's specifications. Endogenous peroxidase activity and nonspecific binding were blocked. Then the sections were incubated with primary antibodies against HMGB1 (1:300; EPR3507; Abcam), RAGE (1:50; 16346‐1‐AP; Proteintech), TLR4 (1:400; GB11519; Servicebio), MMP‐3 (1:200; 17873‐1‐AP, Proteintech), MMP‐9 (1:400; GB11132; Servicebio), MMP‐13 (1:300; GB11247; Servicebio), IL‐1β (1:100; ab9722; Abcam), interleukin‐6 (IL‐6) (1:200; GB11117; Servicebio) and TNF‐α (1:100; ab6671; Abcam) overnight at 4°C in a humidified chamber. The sections were washed with phosphate‐buffered saline (PBS) and incubated with secondary antibodies. Finally, the sections were coloured by reacting with 3,3‐diaminobenzidine (DAB‐0031; Maixin). Haematoxylin was used for counterstaining the structure. Average optical density and positive cells count was determined by researchers who were blinded to the groups using ImageJ.
10
Immunohistochemical Analysis of HMGB1
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Liver sections were deparaffinized, rehydrated and incubated with anti-HMGB1 antibody (1:200, ab18256, Abcam) overnight at 4°C. Subsequently, they were washed and incubated with anti-rabbit IgG (1:1000, ab6721, Abcam) for 30 min at room temperature. The sections were stained with diaminobenzidine (DAB-0031, Maixin Biotechnology), counterstained with hematoxylin and examined under a microscope (Axio Imager M2, Carl Zeiss).
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