The specimens were fixed in 10% neutral w/v phosphate buffer 0.05 M and decalcified in 2% nitric acid and afterward 4% formic acid. Afterward, they were dehydrated with serial ethanol, embedded in paraffin, and cut into 3-μm sections. (FINESSE ME+, Thermo Scientific). The sections were stained with toluidine blue, hematoxylin–eosin and Weigert–Van Gieson stains and viewed under a light microscope. They were blindly scored by two different investigators according to the International Cartilage Repair Society (ICRS) visual histological scale. Immunohistochemistry for collagen type II was performed in all groups, using the EnVision Flex kit and the Autostainer Link (DAKO).
Autostainer link
Manufactured by Agilent Technologies
Sourced in United States, Sweden, Germany
The Autostainer Link is a lab equipment product from Agilent Technologies. It is designed for automated immunohistochemistry (IHC) and in situ hybridization (ISH) staining of tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Pt link, by Agilent Technologies (4 mentions)
Envision flex, by Agilent Technologies (3 mentions)
Envision flex kit, by Agilent Technologies (2 mentions)
Envision flex target retrieval solution, by Agilent Technologies (2 mentions)
Pannoramic 250 digital scanner, by 3DHISTECH (2 mentions)
Caseviewer, by 3DHISTECH (2 mentions)
Finesse me, by Thermo Fisher Scientific (1 mentions)
Envision flex hrp, by Agilent Technologies (1 mentions)
Sc 74470, by Santa Cruz Biotechnology (1 mentions)
Sigma fast dab tablets, by Merck Group (1 mentions)
Cloneep111, by Agilent Technologies (1 mentions)
Caseviewer software, by 3DHISTECH (1 mentions)
Mab319, by Merck Group (1 mentions)
Ab9260, by Merck Group (1 mentions)
Axio scan z1, by Zeiss (1 mentions)
Neutral buffered formalin, by Merck Group (1 mentions)
Ventana discovery xt, by Roche (1 mentions)
Dab chromogen system, by Agilent Technologies (1 mentions)
Chromomap dab, by Roche (1 mentions)
Buffered formalin, by Merck Group (1 mentions)
15 protocols using autostainer link
1
Histological Analysis of Cartilage Specimens
2
Immunohistochemical Evaluation of PARP1 Expression
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Immunohistochemical evaluation was performed using mouse monoclonal anti-human PARP1 antibody (SC-74470; dilution 1:50; clone B-10, Santa Cruz Biotechnology) on 4-μm-thick paraffin sections mounted on silanized slides (code number S3003; Dako Denmark A/S, Glostrup, Denmark). The sections were then deparaffinized, rehydrated, and subjected to heat-induced epitope unmasking. The pT Link module was applied for this purpose using EnVision™ Target Retrieval Solution (20–40 min incubation at 97 °C). An immunohistochemical test was performed utilizing Autostainer Link and EnVision™ FLEX/HRP (SM802; Dako). Stained human placental tissue was used as a positive control. Negative controls were processed with FLEX Rabbit Negative Control, Ready-to-Use (Agilent DAKO) in place of the primary antibody.
Scoring of PARP1 immunostains was performed using the H-score [(percentage at 1+) × 1 + (percentage at 2+) × 2 + (percentage at 3+) × 3], which integrates the intensity and percentage of positive cells into a combined score. The H-score of 280 was used as a cut-off value for high (H-score > 280) and low PARP1 (H-score ≤ 280) expression [35 ].
Scoring of PARP1 immunostains was performed using the H-score [(percentage at 1+) × 1 + (percentage at 2+) × 2 + (percentage at 3+) × 3], which integrates the intensity and percentage of positive cells into a combined score. The H-score of 280 was used as a cut-off value for high (H-score > 280) and low PARP1 (H-score ≤ 280) expression [35 ].
3
ERG Expression Immunohistochemistry Protocol
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ERG expression was evaluated using a commercial rabbit anti-ERG monoclonal antibody (cloneEP111; Dako, Denmark A/S). Deparaffinization, hydration of the slides, and blocking with pre-antibody solution (20 min) where performed in Dako PT Link (Code PT100/PT101). Then, a protocol template was created. The staining steps and incubation times were pre-programmed into the Autostainer Link software (Dako Autostainer). These were diluting anti-ERG primary antibody (1:50 for 20 min at room temp); applying Poly-HRP anti-rabbit IgG (20 min); applying DAB (20 min, Sigma Fast DAB tablets, Sigma-Aldrich, St. Louis MO); counter staining with EnVision FLEX hematoxylin (5 min); and dehydration, clearing, mounting, and covering.
4
Immunohistochemical Analysis of Depressive Brains
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At day of sacrifice, a depressive monkey and a healthy control were anesthetized with ketamine-HCl and euthanized by intravenous pentobarbital overdose. Formalin-fixed, paraffin-embedded brain sections from the cerebrum were obtained; 4 microns sections were processed and stained with hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). For IHC, sections were secured using an automated system, the Dako Autostainer Link. Formalin-fixed paraffin sections were rehydrated with water. Heat-induced epitope retrieval was performed with the FLEX TRS High-pH Retrieval Buffer for 20 min. After peroxidase blocking, the specific monoclonal antibody (IHC-plus CNR1/CB1 pAb, Lifespan, LS-B8253; anti-NUCB1 Rabbit pAb, ABclonal, A3994) was applied at room temperature for 20 min. The FLEX + Rabbit EnVision System was used for detection. DAB chromogen was then applied for 10 min. Slides were counterstained with Mayers hematoxylin for 5 s and then dehydrated and coverslipped. Images were then processed and analyzed using CaseViewer software (2.1 v, 3D Histech Ltd., Budapest, Hungary). Negative controls were included in the run.
5
Xenograft Tumor Analysis and Quantification
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At the end of the experiments, all xenografts were harvested and processed for microscopic examination. The morphology of formalin-fixed and paraffin-embedded xenografts was examined after hematoxylin-eosin and Masson’s trichrome staining. For immunohistochemical analysis, sections were placed on positively charged glass slides and treated with EnVision™ FLEX Target Retrieval Solution (high pH) using a PT-Link (Dako, Denmark). Immunohistochemical staining was performed in an Autostainer Link using EnVision™ FLEX according to the manufacturer’s instructions (Dako). Positive and negative controls were included in each run. Primary antibodies to chromogranin A (MAB319; Merck Millipore, Germany), synaptophysin (Sy38, Dako), Ki67 (AB9260; Merck Millipore), and BAX (B-9; sc-7480; Santa Cruz Biotechnology, CA) were used.
The effects of 177Lu-octreotate on xenografted tumors were evaluated morphologically and graded according to Becket et al. [27 (link)]. The percentage of Ki67-positive tumor cell nuclei was calculated by manual counting of printed images from “hot spots,” including >500 tumor cell nuclei. The intensity of BAX staining was evaluated by two independent observers and graded as absent, weak, moderate, or strong.
The effects of 177Lu-octreotate on xenografted tumors were evaluated morphologically and graded according to Becket et al. [27 (link)]. The percentage of Ki67-positive tumor cell nuclei was calculated by manual counting of printed images from “hot spots,” including >500 tumor cell nuclei. The intensity of BAX staining was evaluated by two independent observers and graded as absent, weak, moderate, or strong.
6
Automated Immunohistochemistry Staining Protocol
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Immunohistochemistry (IHC) was done in automated system using the Dako Autostainer Link. Formalin fixed, paraffin sections were cut at 4 μm and rehydrated to water. Heat induced epitope retrieval was performed with FLEX TRS High pH Retrieval buffer for 20 min. After peroxidase blocking, the specific monoclonal antibodies were applied at room temperature for 20 min. The FLEX + Rabbit EnVision System was used for detection. DAB chromogen was then applied for 10 min. Slides were counterstained with Mayers hematoxylin for 5 s and then dehydrated and coverslipped. Slides were scanned on a Pannoramic 250 digital scanner (3D HISTECH Ltd., Budapest, Hungary) and images scored using the software program ‘Case viewer’ (3D HISTECH Ltd., Budapest, Hungary). Negative controls were included in each staining run.
7
Immunohistochemistry Analysis of Mouse Embryos
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The IHC analyses were performed in collaboration with the Histopathology Core Unit at the Spanish National Cancer Research Centre (CNIO, Madrid, Spain). Mouse embryos were fixed in 4% neutral buffered formalin (Sigma-Aldrich), paraffin embedded, and processed according to standard procedures. For different staining methods, slides were deparaffinized in xylene and samples were rehydrated through graded concentrations of ethanol in water. Consecutive 3-μm sections were stained with H&E, and for IHC, an automated immunostaining platform was used (Ventana Discovery XT, Roche Diagnostics, or Autostainer Link, Dako). Antigen retrieval was performed with high or low (CC1m or RibCC, Roche; PICH, Dako) pH buffer, depending on the primary antibody, and endogenous peroxidase was blocked using 3% hydrogen peroxide. The primary antibodies used were γH2AX (JBV301; 1/25,000; MilliporeSigma, 05-636), p53 (POE316; 1/100; Monoclonal Core Unit CNIO, AM [POE316]), cleaved caspase-3 (1/750; Cell Signaling Technology, 9661) and PICH (D4G8; 1/50; Cell Signaling Technology, 8886). IHC reaction was developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB) (Chromomap DAB, Ventana, Roche; DAB+ Chromogen System, Dako). Nuclei were counterstained with Carazzi’s hematoxylin. Images were acquired using a slide scanner (AxioScan Z1, Zeiss).
8
Immunohistochemical Analysis of PD-L1 Expression
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For histological analyses, tissues were fixed in 10% buffered formalin (Sigma‐Aldrich) and embedded in paraffin. Immunohistochemical staining with anti‐PD‐L1 antibody (rabbit monoclonal antibody (E1L3N); Cell Signaling #13684) was performed on 2.5‐μm tissue sections. Immunohistochemistry was performed using an automated protocol developed for the Autostainer Link automated slide staining system (DAKO, Agilent, Santa Clara, CA, USA). All steps were performed on this staining platform using validated reagents, including deparaffinization, antigen retrieval (cell conditioning), and antibody incubation and detection. Corresponding stainings were acquired and digitalized using the AxioScan.Z1 system (Zeiss). Digitalized images were automatically analyzed with the axiovision version 4.6.2 software (Zeiss). The percentage of PD‐L1 positivity was considered as ratio of PD‐L1‐positive cells to total number of cells.
9
Immunohistochemistry Staining of FFPE Tissues
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FFPE (formalin-fixed paraffin-embedded) tissues were cut to 4 μm sections, placed on slides and deparaffinized. Slides were dried at 60°C and then incubated in FLEX Target Retrieval Solution (TRS) at an optimized pH (high or low). Staining was performed for CD38 (Cell Signaling E7Z8C; 1:100, TRS-high pH), CD16 (Cell Signaling D1N9L; 1:500, TRS-low pH), CD56 (Agilent 123C3; TRS-high pH), and CD14 (Cell Signaling D7A22T; 1:250, TRS-low pH) on a Dako Autostainer Link. Slides were washed, dehydrated, mounted, and scanned on a Leica Biosystem Aperio CS2 light microscope.
10
Automated IHC Staining for p16
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Immunohistochemistry (IHC) for p16 was done using an automated system, the Dako Autostainer Link. Formalin-fixed paraffin sections were cut at 4 microns and rehydrated with water. Heat-induced epitope retrieval was performed with the FLEX TRS High-pH Retrieval Buffer for 20 minutes. After peroxidase blocking, the specific monoclonal antibody (source and dilution: p16 clone G175-405 from BD, USA and diluted 1/25) was applied at room temperature for 20 minutes. The FLEX + Rabbit EnVision System was used for detection. DAB chromogen was then applied for 10 minutes. Slides were counterstained with Mayers hematoxylin for 5 seconds and then dehydrated and coverslipped. Slides were scanned on a Pannoramic 250 digital scanner (3D HISTECH Ltd., Budapest, Hungary) and images scored using the software program “CaseViewer” (3D HISTECH Ltd., Budapest, Hungary). Negative controls were included in the run.
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