Tumors obtained from A549 and H446 xenograft mice were fixed in 4% paraformaldehyde, then were paraffin-embedded and sectioned. Tumor slides were incubated with primary antibodies at 4 °C overnight, incubated with a secondary antibody, and then the chromogenic reaction was conducted with 3, 3-diaminobenzidine (Solarbio, DA1015) and counterstained with hematoxylin (Solarbio, G1080, Beijing China) according to the corresponding manufacturer’s instructions. Images were captured using the Nikon microscope.
G1080
Manufactured by Solarbio
Sourced in China
G1080 is a power supply designed for gel electrophoresis applications. It provides a constant voltage or constant current output to power various electrophoresis systems. The device features a digital display for monitoring output parameters.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Da1015, by Solarbio (2 mentions)
Ar1022, by Boster Bio (2 mentions)
Ar0033, by Boster Bio (2 mentions)
Mk1748, by Boster Bio (2 mentions)
Ab15580, by Abcam (2 mentions)
Rm2235, by Leica (2 mentions)
G1100 100, by Solarbio (2 mentions)
Inverted phase contrast microscope, by Olympus (1 mentions)
Dab kit, by Wuhan Boster Biological Technology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Goat serum, by Solarbio (1 mentions)
Fluoromount g mounting medium, by Southern Biotech (1 mentions)
Alexa fluor 488 555 647, by Thermo Fisher Scientific (1 mentions)
E1170, by Solarbio (1 mentions)
S8032, by Merck Group (1 mentions)
Atg7 rabbit polyclonal antibody, by Thermo Fisher Scientific (1 mentions)
Prestin goat polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Evos fl auto 2, by Thermo Fisher Scientific (1 mentions)
Ab92552, by Abcam (1 mentions)
15 protocols using g1080
1
Immunohistochemical Analysis of Xenograft Tumors
2
Collagen Expression Quantification in Cells
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Immunohistochemistry was performed to analyze the expression levels of collagen type I α1 chain (COL1A1) and collagen type II α1 chain (COL2A1). After 7, 14 and 21 days, cells were washed three times with PBS, fixed with 95% alcohol at room temperature for 30 min and treated with Triton X-100 (Sigma-Aldrich; Merck KGaA) at room temperature for 10 min. To eliminate endogenous peroxidase activity, cells were incubated with 3% H2O2 at room temperature for 15 min and blocked with goat serum (Beijing Solarbio Science & Technology Co., Ltd.) for 15 min at room temperature. After incubating with COL1A1 (1:200; Wuhan Boster Biological Technology, Ltd., Wuhan, China) and COL2A1 (BA0533; 1:200; Wuhan Boster Biological Technology Ltd.) primary antibodies overnight at 4°C, cells were incubated with the secondary antibody (G1080, 1:200; Beijing Solarbio Science & Technology Co., Ltd.) and followed by biotin-labeled horseradish peroxidase (Zhongshanjinqiao Biotechnology Inc., Wuhan, China) at room temperature for 15 min. A DAB kit (Wuhan Boster Biological Technology, Ltd.) was used to visualize antibody binding. Finally, images of the cells were captured under an inverted phase contrast microscope (Olympus Corporation).
3
In Situ Hybridization of ARC/Arg3.1 mRNA
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Paraffin sections were dewaxed in water and digested with protease K (20 μl/ml) at 37℃ for 30 min. The 3% methanol-hydrogen peroxide was added, and the slide was placed in phosphate buffer saline (pH 7.4) (Boster Biological Technology Co., Ltd., China, AR0033) to block endogenous peroxidase. After pre-hybridization, ARC/Arg3.1 mRNA probe (5`-CGCTG GGTCA AGCGT GAGAT GCACG TGTGG AGGGA-3`; 5`-TATTG GCTGT CCCAG ATCCA GAACC ACATG AATGG-3`; 5`-TGGCG TAAGC GGGAC CTGTA CCAGA CACTG TATGT-3`) hybridization solution containing the probe was added (Boster Biological Technology Co., Ltd., China, MK1612) at a concentration of 20 μl. Hybridization was conducted at 37℃ in an incubator overnight, and then the hybridization solution was washed away. BSA blocking solution was then added, followed by a drop of mouse anti-digoxigenin-labeled peroxidase (Boster Biological Technology Co., Ltd., China, MK1748). DAB (Boster Biological Technology Co., Ltd., China, AR1022) was used to show color, and positive results ranged from yellow to brownish yellow. The nucleus of hematoxylin staining (Beijing Solarbio Science & Technology Co., Ltd., China, Ltd, G1080) was blue. The tissue was then dehydrated, and microscopic examination, image acquisition, and analysis were performed. Three fields of view were randomly selected for each slice for statistical analysis.
4
Immunofluorescence Analysis of Cochlear Structures
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Isolated cochleae were immersed in 4% paraformaldehyde and then decalcified with 0.5 M EDTA (Solarbio, E1170). For cryosectioning, cochleae were immersed in increasing concentrations of 10–30% (w/v) sucrose (Biosharp, Amresco 0335) and then with serial mixtures of OCT (Sakura, Tissue-Tek 4583) and sucrose. The sections and whole mounts were blocked with phosphate-buffered saline blocking solution containing 5% donkey serum, 1% bovine serum albumin, 0.02% sodium azide (Sigma-Aldrich, S8032), and 0.5% Triton; incubated with diluted primary antibodies; and further with fluorescence-conjugated secondary antibodies (Alexa Fluor 488/555/647, Invitrogen). The primary antibodies were Atg7 rabbit polyclonal antibody (Thermo Fisher, PA5-35203, 1:300); Myosin VIIa mouse polyclonal antibody (Thermo Fisher, PA1-936, 1:500); Prestin goat polyclonal antibody (Santa Cruz, sc-22694, 1:200); CtBP2 mouse monoclonal antibody (BD Biosciences, 612044, 1:200); and P62 Guinea Pig polyclonal antibody (Progen, GP62-C, 1:200). The anti-fade Fluoromount-G mounting medium (SouthernBiotech, 0100-01) was used for mounting. The fluorescence images were obtained by a Zeiss LSM 710 confocal microscope. For hematoxylin staining, the whole mounts were stained with diluted hematoxylin (Solarbio, G1080) for 5 min.
5
Immunohistochemical Analysis of Protein Targets
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The frozen tissue sections were fixed in pre-cooled formaldehyde fixative solution (85 mM Na2HPO4, 75 mM KH2PO4, 4% paraformaldehyde, pH 6.4) for 10 min (4 °C) and washed three times for 5 min for PBS. Then, the sections were incubated in 0.3% H2O2 in methanol at room temperature for 10 min to block endogenous peroxidase activity, and then washed three times for 5 min with PBS. After incubating the tissue sections with blocking buffer (5% BSA in PBS) for 1 h at room temperature, the sections were incubated with the BLM antibody (Bioss, bs-12872R) or the P21 antibody (Bioss, bs-0741R) diluted in blocking buffer (1:50 or 1:200) at 4 °C, overnight. After incubation with the secondary antibody diluted in blocking buffer for 1 h (1:100), the sections were washed three times for 5 min with PBS and the DAB substrate solution (Thermo Scientific, #34002) was applied for 5 min. The slides were counterstained by immersing the slides in hematoxylin (Solarbio, G1080) for 15 s, and then rinsing them in tap water for 15 min before mounting. Images were taken under the Evos FL Auto 2 (Thermo).
6
Immunohistochemical Analysis of Tumor Tissues
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Tumor tissues were subjected to antigen retrieval for 20 min and probed at 4 °C overnight with primary antibodies including rabbit anti-PCNA (ab92552, 1:200, Abcam, Cambridge, MA) and rabbit anti-Ki67 (ab15580, 1:200, Abcam). After that, biotinylated secondary antibody (goat anti-rabbit, ab150077, 1:500, Abcam) was added for further incubation for 30 min. Then, DAB (Solarbio, Beijing, China, DA1015) was added for development and hematoxylin (Solarbio, G1080) was used for counterstaining. The sections were dehydrated, cleared, and sealed before microscopic observation in five representative fields.
7
In Situ Hybridization of Egr-1 mRNA in Paraffin Sections
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Paraffin sections were dewaxed in water and boiled in repair solution for 10 min. After natural cooling, digested with protease K (20 μl/ml) at 37 °C for 30 min. Then 3% methanol-hydrogen peroxide was added, and the slide was placed in phosphate buffer saline (pH 7.4) (Boster Biological Technology Co., Ltd., China, AR0033) to block endogenous peroxidase. After pre-hybridization, Egr-1 mRNA probe (5`-GAGGA GATGA TGCTG CTGAG CAACG GGGCT-3`; 5`-GCCTT TGCCA CTCAG TCGGG CTCCC AGGAG-3`; 5`-CCTTT TCTCC CAGCA CAATT GAAAT TTGCT-3`) hybridization solution containing the probe was added (Boster Biological Technology Co., Ltd., China, MK1748) at concentration of 20 μl. Hybridization was conducted at 37 °C in an incubator overnight, and then the hybridization solution was washed away. BSA blocking solution was then added, followed by a drop of mouse anti-digoxigenin-labelled peroxidase (Boster Biological Technology Co., Ltd., China, MK1748). DAB (Boster Biological Technology Co., Ltd., China, AR1022) was used to show colour, and positive results ranged from yellow to brownish yellow. The nucleus of haematoxylin staining (Beijing Solarbio Science & Technology Co., Ltd., China, Ltd., G1080) was blue and sealed via dehydration. Chemiluminescence was measured using image analysis by Image-Pro Plus.
8
Immunohistochemistry Analysis of Tumor Tissues
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For IHC analysis, samples were collected and paraformaldehyde fixed, paraffin-embedded sections of tumor tissues (5 μm thick) were mounted on slides coated with 2-aminopropyltriethoxysilane, which then were baked, deparaffinized, rinsed with 3% hydrogen peroxide. After that, these sections were washed and then blocked with 3% goat serum for 5 min and subsequently incubated with an anti-HIBCH (Abcam, ab153826, Cambridge, UK; diluted 1:500) or PCNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-56; diluted 1:100) antibody at 4 °C overnight. Finally, the following steps were in accordance to the protocols provided by the manufacturer of GTVisionTMIII Complex (Gene Tech, GK500705, Shanghai, China). The nucleus was stained with hematoxylin (Solarbio, G1080, Beijing, China). Sections were further mounted with neutral gums. IHC sections were photographed by Mantra 1.01 (Perkin Elmer, Waltham, MA, USA).
9
Paraffin-embedded Tissue Immunohistochemistry
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Tissues from main organs were immediately fixed in 4% PFA and then embedded in paraffin. Sections (5 μm) were stained with HE according to standard methods. For IHC staining, the sections were baked at 60 °C for 30 min and were then deparaffinized by xylene, absolute ethanol, 75% ethanol, and boiled in citrate buffer for antigen retrieval. Then, the sections were incubated with antibodies to CD4 (ab183685, Abcam, diluted 1:500) or to CD8 (98941S, CST, diluted 1:500). Samples were counterstained nucleus with hematoxylin (G1080, Solarbio, China).
10
Immunohistochemical Analysis of Tumor Markers
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Tumor tissues were fixed in 4% paraformaldehyde, and dehydrated with gradient ethanol. Then, tissues were embedded into paraffin (YA0011, Solarbio) and cut into sections with a thickness of 5 μm. The restoration was executed with sodium citrate buffer (pH 6.0, P0081, Beyotime) at 94 °C for 15 min. Subsequently, sections were sealed with 1% bovine serum albumin (BSA, ST2249, Beyotime) for one hour, and hatched with primary antibodies targeting Ki-67 (1:200, ab15580, Abcam), FOXM1 (1:250, ab207298, Abcam) and STMN1 (1:2000, ab52630, Abcam) overnight at 4 °C. The secondary antibody HRP labeled anti-rabbit IgG antibody (ab288151, Abcam) was used to incubate with sections at 37 °C for 30 min. The sections were re-stained with hematoxylin (G1080, Solarbio), and pictured under a light microscope (Olympus).
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