To investigate the distribution of collagen fiber, Masson trichrome staining kit (D026; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was applied on lacrimal gland tissues. In brief, paraffin sections were dewaxed before staining. Tissues were rinsed with double distilled water for two minutes at 37°C, followed by nuclear staining, cytoplasm staining, color separation, and counterstaining according to the manufacturer's instructions. After douching with pure ethyl alcohol, sections were mounted with mounting medium (H-5000; Vector Laboratories, Burlingame, CA, USA) and observed under a light microscope.
H 5000
Manufactured by Vector Laboratories
Sourced in United States
The H-5000 is a high-performance laboratory equipment designed for precision research and analysis. It offers a core function of providing accurate and consistent measurements, without further interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Zen blue software, by Zeiss (3 mentions)
H 3401, by Vector Laboratories (3 mentions)
Pk 6102, by Vector Laboratories (3 mentions)
Vectamount permanent mounting medium, by Vector Laboratories (2 mentions)
Eclipse ti u microscope, by Nikon (2 mentions)
S3022, by Agilent Technologies (2 mentions)
Sp 6000, by Vector Laboratories (2 mentions)
Sk 4100, by Vector Laboratories (2 mentions)
Ab150681, by Abcam (2 mentions)
Axio scan z1 slide scanner, by Zeiss (2 mentions)
Abc kit, by Vector Laboratories (2 mentions)
Mom blocking buffer, by Vector Laboratories (2 mentions)
Citrate buffer, by Vector Laboratories (2 mentions)
Masson trichrome staining kit, by Nanjing Jiancheng (1 mentions)
Mca711g, by Bio-Rad (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
S 2012, by Vector Laboratories (1 mentions)
Ba 1000, by Vector Laboratories (1 mentions)
Axiocam hrc camera, by Zeiss (1 mentions)
Axioimager microscope, by Zeiss (1 mentions)
23 protocols using h 5000
1
Collagen Fiber Distribution in Lacrimal Gland
2
Immunohistochemical Staining of CD11b+ Cells
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PFA-fixed brain sections were washed twice with DPBS (without calcium and magnesium) before undergoing fixation with 95% ethanol (pre-chilled to 4 °C) for 10 min. Antigen retrieval was performed with a 15-min incubation of pre-warmed (37 °C) trypsin (0.25%) before blocking endogenous peroxidase activity with a 5-min incubation of 0.3% hydrogen peroxide and 0.3% normal rabbit serum in DPBS. Sections were blocked in DPBS with 1.5% normal rabbit serum for 20 min before primary antibody incubation with 1:100 rat anti-mouse CD11b (Bio-Rad MCA711G, Hercules, CA, USA) in DPBS with 2.5% normal rabbit serum. The primary antibody was visualized with the avidin-biotin horseradish peroxidase technique in accordance with the manufacturer’s instructions (Vector Laboratories, Vectastain Elite Kit PK6104, NovaRED Peroxidase (HRP) Substrate Kit SK-4800, Burlingame, CA, USA). Sections were then counterstained with hematoxylin in accordance with the manufacturer’s instructions (Vector Laboratories, H-3401, Burlingame, CA, USA), and mounted with Vectamount Permanent Mounting Medium (Vector Laboratories, H-5000, Burlingame, CA, USA). Brightfield images were subsequently obtained using a Zeiss Axio Scan.Z1 slide scanner with the ZenBlue software (Carl Zeiss, Jena, Germany).
3
Immunohistochemical Staining Protocol
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Immunohistochemical staining was performed following the protocols indicated in a previous study by Jo et al.48 (link) Briefly, the tissue slides were fixed with 100% cold acetone for 10 min and washed with flowing water for 10 min to remove the OCT compound (SAKURA, 4583). After washing with 1x PBS three times, tissue slides were incubated with 1x PBS including 0.3% Triton X-100 for 10 min and blocked with BLOXALL (Vector Lab, SP-6000) for 30 min at room temperature. The tissue slides were immunostained with primary antibody (COL1A1, Santa, 8784; 1:100 or YAP, ABclonal, A1002; 1:200) diluted with antibody diluent (DAKO, S3022) at room temperature for 1 h. After washing three times with 1x PBST, the tissue slides were incubated with biotinylated anti-rabbit IgG secondary antibodies (Vector Lab, BA-1000; 1:200) mixed with one drop of normal horse serum (Vector Lab, S-2012) for 1h. Consecutively, tissue slides were incubated with an ABC kit (Vector Lab, PK-6102) for 30 min and stained with a DAB substrate kit (Vector Lab, sk4100) for 1–5 min. Afterward, tissue slides were stained with hematoxylin (Merck, 1.05174.0500) for 10 s and dehydrated with gradually increasing ratios of ethanol solution (50–100% ethanol). Finally, tissue slides were mounted with a permanent mounting medium (Vector Lab, H-5000). Images were gathered with a Nikon Eclipse Ti-U microscope.
4
Toluidine Blue Staining of Mouse Brain
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The paraffin blocks were prepared by Division of Human Pathology of Michigan State University. Briefly, mouse brain sections were dewaxed in xylene and rehydrated in alcohol. Then, sections were stained in toluidine buffer [1 g toluidine blue (Sigma) in 100 mL 95% ethanol] at room temperature for 20 min. Quick rinse in tap water and 70% ethanol to remove excess stain. After dehydration and wax, sections were mounted in mounting media H5000 (Vector Laboratories). Images were captured using a Zeiss Axio Imager microscope (Carl Zeiss GmbH, Oberkochen, Germany) and an installed AxioCam HRc camera (Carl Zeiss GmbH) with image acquisition via Zeiss Zen Pro software (v.2.3; Carl Zeiss GmbH).
5
Immunohistochemical Analysis of Liver Tissue
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The methods are described in detail in the Supporting Information. Liver sections were deparaffinized in EZ‐DeWax (HK 585‐5K; BioGenex, San Ramon, CA) and antigen retrieval was performed in antigen retrieval solution (HK 086‐9K; BioGenex). Slides were treated sequentially with peroxidase suppressor, universal block, and avidin (36000; Thermo Fisher Scientific, Waltham, MA). Slides were incubated sequentially with primary antibody diluted in universal block containing a biotin, biotinylated goat antimouse IgG, and avidin‐biotin complex. Slides were developed with Immpact Nova Red peroxidase substrate (SK‐4805; Vector Labs, Burlingame, CA), counterstained with Mayer’s Hematoxylin (S3309; Agilent Dako, Santa Clara, CA), dehydrated, and mounted in nonaqueous mounting media (H‐5000; VectorLabs). Ki67 was detected with a mouse monoclonal antibody (M7240, MIB‐1; Agilent Dako) at 1:100 dilution and activated caspase 3 was detected with a rabbit antibody (9664S, 5A1E; Cell Signaling Technology, Danvers, MA) at 1:100 dilution.
6
Histological Analysis of Tendon Allografts
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Cryo-sectioned slides at 6-μm thickness were used for histological analysis. The staining procedures were conducted following the standard protocols indicated in the manufacturer’s instructions. Hematoxylin and eosin (H&E) stain was used for histologic and cytologic analyses, and Picrosirius red (PSR; Abcam, ab150681) was used for types I and III collagen analysis. All stained slides were mounted using a permanent mounting medium (Vector Labs, H-5000). Images of cells injected into the tendon allografts were observed at 200x magnification using bright-field microscopy, and the number of cells was counted on five consecutive slides. All measurements were analyzed by two independent observers blinded to each other.
7
Immunohistochemical Staining of CD11B
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Brain sections were washed twice with Dulbecco’s phosphate-buffered saline (DPBS) (without calcium and magnesium) before undergoing fixation with 95% ethanol (pre-cooled to 4°C) for 10 min. Antigen retrieval was performed with a 15-min incubation of pre-warmed (37°C) trypsin (0.25%) before blocking endogenous peroxidase activity with a 5-min incubation of 0.3% hydrogen peroxide and 0.3% normal rabbit serum in DPBS. Sections were blocked in DPBS with 1.5% normal rabbit serum for 20 min before primary antibody incubation with 1:100 CD11B (Serotec MCA711G; Serotec, Oxford, United Kingdom) in DPBS with 2.5% normal rabbit serum. The primary antibody was visualized with the avidin–biotin horseradish peroxidase technique in accordance with manufacturer’s instructions [Vector Laboratories, Vectastain Elite Kit PK6104, NovaRED Peroxidase (HRP) Substrate Kit SK-4800, Burlingame, CA, United States]. Sections were then counterstained with hematoxylin in accordance with the manufacturer’s instructions (Vector Laboratories, H-3401, Burlingame, CA, United States), and mounted with Vectamount Permanent Mounting Medium (Vector Laboratories, H-5000, Burlingame, CA, United States). Brightfield images were subsequently obtained using a Zeiss Axio Scan.Z1 slide scanner with the ZenBlue software (Carl Zeiss, Jena, Germany).
8
Immunofluorescence Staining of Human Dental Pulp Cells
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Human dental pulp cells fixed in 4% paraformaldehyde (PFA; T&I, BFA-9020) and deparaffinized tissue sections were treated with phosphate-buffered saline (PBS) containing 0.5% Triton X-100 (Amresco, 0694) for permeabilization. After being washed and blocked, the cells were incubated for 1 h with LC3B antibody (1:100) or α-TUBULIN (1:100, YOL1/34; Santa Cruz Biotechnology, sc-53030) antibody and TAU antibody (1:50) in blocking buffer (PBS and 2% bovine serum albumin (BSA; Gibco BRL, 30063-572)). Tissue sections were incubated with α-TUBULIN antibody (1:200) overnight at 4°C. Subsequently, fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (1:200; Thermo Fisher Scientific, F2765), Alexa Fluor 488-conjugated goat anti-rat IgG antibody (1:200; Thermo Fisher Scientific, A11006), or Cy3-conjugated goat anti-mouse IgG antibody (1:200; Merck Millipore, AP124C) was applied. After being washed, the chromosomal DNA in the nucleus was stained with 4’,6-diamidino-2-phenylindole (DAPI) during the mounting procedure (Vector Labs, H-5000). Samples were visualized using confocal laser scanning microscopy (Carl Zeiss, LSM 800).
9
Immunohistochemical Staining of Tissue Slides
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Tissue slides were baked for 1 hour at 65°C, deparaffinized in xylene, and rehydrated through a series of graded alcohols. After diH2O washes, slides were treated with antigen unmasking solution (1:100; SKU H-3300-250, Vector Laboratories) at 95°C for 12 min according to the manufacturer’s protocol. Hydrogen peroxide, blocking, primary antibody binding, HRP-conjugated secondary antibody, and 3, 3′-diaminobenzidine (DAB) were done using the Mouse and Rabbit Specific HRP/DAB IHC Detection Kit - Micro-polymer (ab236466, Abcam). Before the protein block, tissues were treated with 0.1% Triton X-100 diluted in 1× tris-buffered saline (TBS) for 5 min to unmask the antigens expressed in the nucleus. After overnight primary antibody incubation at 4°C and endogenous treatment with secondary anti-rabbit antibody at room temperature (RT) for 20 min, the staining was visualized with DAB with exposure times synchronized throughout all tissue samples within an antibody group for the exact same time. All slides were counterstained with hematoxylin (Hematoxylin QS, H-3404, Vector Laboratories) for 20 s at RT, dehydrated in ethanol, cleared in xylene, and mounted with VectaMount (permanent mounting medium, H-5000, Vector Laboratories).
10
Immunohistochemical Analysis of CD44 Expression
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Tumor tissues were fixed with 4% paraformaldehyde, and then dehydrated and embedded in paraffin. From the rehydrated tissue slices, antigen retrieval was performed using the heat-induced antigen retrieval in citrate buffer (Vector Laboratories, CA). The tissues were made permeable with PBS, containing 0.2% TritonX-100, and then treated with 3% hydrogen peroxide to inactivate the endogenous peroxidase. The tissues were washed with PBS, containing 0.05% Tween-20, and then incubated for 2 h with a blocking solution (MOM blocking buffer, Vector Laboratories, CA). The samples were then incubated with CD44 antibody overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature. Signals were amplified using an ABC kit (Vector Laboratories) and visualized using a 3,3′-diaminobenzidine substrate kit (SK-4105, Vector Laboratories). The tissues were further stained with H&E, dehydrated, and mounted (H5000, Vector Laboratories).
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