Automated slide stainer

Manufactured by Agilent Technologies

Sourced in Australia

The Automated Slide Stainer is a laboratory instrument designed for automated staining of microscope slides. It performs precise and consistent staining procedures on multiple slides simultaneously, streamlining the sample preparation workflow.

Automatically generated - may contain errors

Lab products found in correlation

Irdye 680 fluorophore, by LI COR (3 mentions) Genepix al4200, by Molecular Devices (3 mentions) Genepix pro 7, by Molecular Devices (3 mentions) Nitrocellulose coated slides, by Grace Bio-Labs (3 mentions) Immpact novared peroxidase substrate, by Vector Laboratories (2 mentions) K4003, by Agilent Technologies (2 mentions) Proteome profiler mouse adipokine array kit, by R&D Systems (1 mentions) Ab66155, by Abcam (1 mentions) Diaminobenzidine, by Agilent Technologies (1 mentions) Ab28364, by Abcam (1 mentions) A0452, by Agilent Technologies (1 mentions) Cst9661, by Cell Signaling Technology (1 mentions) Mayer s hematoxylin, by Merck Group (1 mentions) Pt link module, by Agilent Technologies (1 mentions) Envision rabbit detection system, by Agilent Technologies (1 mentions) Tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions) Anti granzyme b, by Abcam (1 mentions) Nanozoomer xr slide scanner, by Hamamatsu Photonics (1 mentions)

9 protocols using automated slide stainer

Reverse Phase Protein Array Analysis of Adipokines

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Protein lysates were prepared by the BCM Antibody-Based Proteomics Core for reverse phase protein array assays. The Aushon 2470 Arrayer (Aushon BioSystems) with a 40 pin (185 μm) configuration was used to spot lysates onto nitrocellulose-coated slides (Grace Bio-Labs). The slides were probed with 220 antibodies against total and phospho-proteins using an automated slide stainer (Dako). Primary antibody binding was detected using a biotinylated secondary antibody followed by streptavidin-conjugated IRDye 680 fluorophore (LI-COR). Fluorescent-labeled slides were scanned on a GenePix AL4200 and the images were analyzed with GenePix Pro 7.0 (Molecular Devices). Background-subtracted total fluorescence intensities of each spot were normalized for variation in total protein (Sypro Ruby) and nonspecific labeling. For adipokine analysis, tissue lysates from four samples were pooled (125 μg/sample) and incubated with membranes from the Proteome Profiler Mouse Adipokine Array Kit (R&D Systems #ARY013) according to the manufacturer’s instructions.

Cox A.R., Masschelin P.M., Saha P.K., Felix J.B., Sharp R., Lian Z., Xia Y., Chernis N., Bader D.A., Kim K.H., Li X., Yoshino J., Li X., Li G., Sun Z., Wu H., Coarfa C., Moore D.D., Klein S., Sun K, & Hartig S.M. (2022). The rheumatoid arthritis drug auranofin lowers leptin levels and exerts anti-diabetic effects in obese mice. Cell metabolism, 34(12), 1932-1946.e7.

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Immunohistochemical Analysis of Cellular Markers

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Tissues were fixed in formalin for 24 hours and paraffin-embedded. Four micron sections were deparaffinized and antigen retrieval was performed with either citrate buffer, pH 6 (for Ki67) or Tris-EDTA buffer, pH 9 (for caspase-3, CD3 and CD31). Sections were boiled in retrieval buffer for 20 minutes, then cooled for 20 minutes. Using an automated slide stainer (Dako Australia, North Sydney, NSW, Australia) sections were quenched with hydrogen peroxide, incubated with primary antibody then secondary antibody (K4003, Dako) and revealed with diaminobenzidine (Dako) or ImmPact NovaRed Peroxidase Substrate (Vector Laboratories, Burlingame, CA). Sections were counterstained in Mayer's hematoxylin. Primary antibodies were: Ki67 (ab66155, Abcam, final dilution 1:250), cleaved caspase-3 (CST9661, Cell Signaling Technology, Beverly, MA, final dilution 1:300), CD3 (A0452, Dako, final dilution 1:400) and CD31 (ab28364, Abcam, final dilution 1:100). Images were analyzed using CellProfiler, Broad Institute, MA [59 (link)], quantifying the number of positively stained pixels or total number of cells, identified by hematoxylin staining and the number of diaminobenzidine- or ImmPact NovaRed-positive cells present in the images. See Supplementary Methods for further details.

Scully T., Firth S.M., Scott C.D., de Silva H.C., Pintar J.E., Chan-Ling T., Twigg S.M, & Baxter R.C. (2016). Insulin-like growth factor binding protein-3 links obesity and breast cancer progression. Oncotarget, 7(34), 55491-55505.

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Immunohistochemical Detection of TOX3 in Breast Cancer

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Immunohistochemical detection of TOX3 was performed on 4-μm sections of formalin fixed paraffin embedded tissue stained with AJ-33. Staining was done using an automated slide stainer (Dako, Carpinteria, California, USA). Antigen retrieval was performed using the Dako PT Link Module and low pH buffer. Staining was visualized using the Dako Envision + Rabbit Detection System. Slides were subsequently counterstained with Mayer’s hematoxylin (Sigma-Aldrich). Three commercially available tissue arrays (Pantomics, Richmond, CA, USA) that contained a total of 210 primary breast cancer samples in duplicate were analyzed for TOX3 expression. Some samples were destroyed during the processing and therefore removed from analysis. Thus, the TOX3 histological data shown here are derived from a total of 188 breast tumors. Other histological data that were associated with individual tumors on the tissue array were supplied by the manufacturer. For some histological analysis, the Department of Pathology and Laboratory Medicine at CSMC supplied normal breast tissue obtained following mammoplasty. Samples were obtained under a waiver of consent and provided in an anonymous fashion, so that the connection to individual patients was destroyed prior to their analysis. This work was performed under Cedars-Sinai Medical Center’s Institutional Review Board Study Number: Pro 00033387.

Seksenyan A., Kadavallore A., Walts A.E., de la Torre B., Berel D., Strom S.P., Aliahmad P., Funari V.A, & Kaye J. (2015). TOX3 is expressed in mammary ER+ epithelial cells and regulates ER target genes in luminal breast cancer. BMC Cancer, 15, 22.

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Quantitative Assessment of Vitamin D Receptor Expression

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From each TMA block, a 5-micron section was cut and used for immunohistochemical (IHC) staining for VDR with a validated monoclonal antibody 9A7 (ThermoFisher catalog # MA1-710, Waltham, MA) and a Dako automated slide stainer following standardized protocols established by PRN. The antibody showed an exclusive nuclear staining without cytoplasmic reactivity in breast tissues (Figure 1), which is consistent with the literature (10 (link), 24 (link)). After staining, whole-slide digital images were captured by the Aperio ScanScope CS Slide Scanner, and a computer-assisted image analysis algorithm optimized for the VDR antibody was used for automated quantitative assessment of staining intensity and percent of positive staining area. Any tumor with a total nuclei count less than 15 was excluded (n=18) from the analysis. An immunoreactive score (IRS) was computed as the product of intensity score (0-3) and percent of positive nuclei score (0-4) for each core and scores across multiple cores of each tumor block were compiled into a final score by the average score weighted by the total count of nuclei of each core. The resultant score thus ranged from 0-12. Based on the distribution of the IRS, VDR expression was classified into three levels with largely similar number of cases in each level: low (0-2), moderate (3-5), and strong (6-12) (see Figure 1 for representative stains).

Al-Azhri J., Zhang Y., Bshara W., Zirpoli G., McCann S.E., Khoury T., Morrison C.D., Edge S.B., Ambrosone C.B, & Yao S. (2016). Tumor Expression of Vitamin D Receptor and Breast Cancer Histopathological Characteristics and Prognosis. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(1), 97-103.

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Reverse Phase Protein Array Analysis

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Protein lysates were prepared by the BCM Antibody-Based Proteomics Core for reverse phase protein array assays. The Aushon 2470 Arrayer (Aushon BioSystems) with a 40 pin (185 μm) configuration was used to spot lysates onto nitrocellulose-coated slides (Grace Bio-Labs). The slides were probed with >220 antibodies against total and phospho-proteins using an automated slide stainer (Dako). Primary antibody binding was detected using a biotinylated secondary antibody followed by streptavidin-conjugated IRDye 680 fluorophore (LI-COR). Fluorescent-labeled slides were scanned on a GenePix AL4200 and the images were analyzed with GenePix Pro 7.0 (Molecular Devices). Background-subtracted total fluorescence intensities of each spot were normalized for variation in total protein (Sypro Ruby) and nonspecific labeling.

Felix J.B., Saha P.K., de Groot E., Tan L., Sharp R., Anaya E.S., Li Y., Quang H., Saidi N., Abushamat L., Ballantyne C.M., Amos C.I., Lorenzi P.L., Klein S., Gao X, & Hartig S.M. (2024). N-acetylaspartate from fat cells regulates postprandial body temperature. Research Square.

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Multiplexed Protein Profiling of Cell Lysates

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Protein lysates were prepared with Tissue Protein Extraction Reagent (Thermo Fisher) containing protease and phosphatase inhibitors (Roche). The Aushon 2470 Arrayer (Aushon BioSystems) with a 40 pin (185 μm) configuration was used to spot lysates onto nitrocellulose-coated slides (Grace Bio-Labs). The slides were probed with 220 antibodies against total and phosphoprotein proteins using an automated slide stainer (Dako). Primary antibody binding was detected using a biotinylated secondary antibody followed by streptavidin-conjugated IRDye 680 fluorophore (LI-COR Biosciences). Fluorescent-labeled slides were scanned on a GenePix AL4200, and the images were analyzed with GenePix Pro 7.0 (Molecular Devices). Total fluorescence intensities of each spot were normalized for variation in total protein (Sypro Ruby) and nonspecific labeling.

Koh E.H., Chernis N., Saha P.K., Xiao L., Bader D.A., Zhu B., Rajapakshe K., Hamilton M.P., Liu X., Perera D., Chen X., York B., Trauner M., Coarfa C., Bajaj M., Moore D.D., Deng T., McGuire S.E, & Hartig S.M. (2018). miR-30a Remodels Subcutaneous Adipose Tissue Inflammation to Improve Insulin Sensitivity in Obesity. Diabetes, 67(12), 2541-2553.

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Multiplexed Antibody Profiling of Cell Lysates

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After DS treatment for 24 hours, cells were collected and lysed in lysis buffer. All samples were diluted to a final concentration of 1 mg/mL, and then 30 μL of each sample, arrayed in a series of dilutions, was printed in duplicate on slides. The slides were then subjected to immunostaining with a panel of 207 commercially available antibodies¹³. Slides were stained on an automated slide stainer (DAKO, Carpinteria, CA) using biotin-linked peroxidase-catalyzed signal amplification.

Koul D., Wang S., Wu S., Saito N., Zheng S., Gao F., Kaul I., Setoguchi M., Nakayama K., Koyama K., Shiose Y., Sulman E.P., Hirota Y, & Yung W.K. (2017). Preclinical therapeutic efficacy of a novel blood-brain barrier-penetrant dual PI3K/mTOR inhibitor with preferential response in PI3K/PTEN mutant glioma. Oncotarget, 8(13), 21741-21753.

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Immunohistochemical Quantification of CD8+ and Granzyme B+ Cells

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Tissues were fixed in formalin for 24 h and paraffinembedded. Four-micrometre sections were deparaffinised and antigen retrieval was performed using a water bath with either citrate buffer, pH 6 (for granzyme B) or Tris-EDTA buffer, pH 9 (CD8a). Sections were boiled in retrieval buffer for 20 min, and then cooled for 20 min. Using an automated slide stainer (Dako Australia), sections were quenched with hydrogen peroxide, incubated with primary antibody and then secondary antibody (K4003, Dako) and revealed with ImmPact NovaRed Peroxidase Substrate (Vector Laboratories, Burlingame, CA, USA). Sections were counterstained in Mayer's haematoxylin. Primary antibodies used were anti-CD8α (#14-0808, Affymetrix eBioscience, 1 μg/mL) and anti-Granzyme B (#ab4059, Abcam, 1.8 μg/mL). Images were analysed using CellProfiler software (Broad Institute, MA, USA) as previously described (Scully et al. 2016) (link). The data are presented as the number of positively stained cells as a proportion of the total number of cells present in the fields of view examined.

Scully T., Scott C.D., Firth S.M., Sedger L.M., Pintar J.E., Twigg S.M, & Baxter R.C. (2018). Enhancement of mammary tumour growth by IGFBP-3 involves impaired T cell accumulation. Endocrine-related cancer, 25(2).

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Immunohistochemistry for TIGAR and Alpha-Synuclein

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Immunohistochemistry using a standard avidin-biotin complex (ABC) method was performed on 4m-thick, formalin-fixed, paraffin-embedded sections. Details of the antisera as well as antigen retrieval and immunohistochemistry conditions are detailed in supplementary table S1. TIGAR Immunostaining was performed with the IntelliPATH FLX Detection Kit and autostainer system (Menarini Diagnostics). For p53, slides were stained on the Dako Omnis Automated Slide Stainer using the Dako Envision Flex High pH (GV80011) kit. The whole slide images were captured using a Hamamatsu NanoZoomer XR slide scanner. Double labelling immunohistochemistry was attempted to investigate co localisation of TIGAR with alpha-synuclein. Two formats were attempted: 1) anti alphasynuclein antibody with Alexa Fluor 488 or 555 and TIGAR labelled with DAB or 2) both alpha-synuclein and TIGAR in fluorescence. Both formats involved incubation with Sudan Black to mask auto fluorescent material.
To further confirm immunostaining of TIGAR in Lewy bodies, adjacent sections were immunostained for TIGAR using the PA5-29152 (Thermo Scientific) antibody and alpha-synuclein and digitised. The resultant whole slide images aligned such that the same structures could be assessed in the adjacent sections.

López K.L.R., Simpson J.E., Watson L.C., Mortiboys H., Hautbergue G.M., Bandmann O, & Highley J.R. (2019). TIGAR inclusion pathology is specific for Lewy body diseases. Brain research, 1706.

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