Tissue was fixed and embedded in paraffin blocks. Serial 4-µm paraffin sections were deparaffinized by EZ prep (Ventana Medical Systems, Tucson, AZ) and subjected to a 64-min pre-treatment using Cell Condition 1 solution (Ventana Medical Systems, Tucson, AZ). The slides were incubated with primary antibodies for 32 min using the automated Ventana Benchmark XT (Ventana Medical Systems, Tucson, AZ). Signals were detected with the Optiview DAB Detection Kit (Ventana Medical Systems, Tucson, AZ) according to the manufacturer’s protocol on an Olympus BX53 microscope (Olympus Corporation, Tokyo, Japan). All sections were counterstained with haematoxylin (Ventana Medical Systems, Tucson, AZ). Antibodies against GROα (Santa Cruz Biotechnology, Dallas, TX) are listed in Supplementary Table S1 .
Haematoxylin
Manufactured by Roche
Haematoxylin is a natural dye derived from the Logwood tree. It is a common staining agent used in histology and pathology laboratories for the visualization of cellular structures in tissue samples. Haematoxylin stains nuclei blue-purple, providing contrast for the identification of cells and their components.
Automatically generated - may contain errors
Lab products found in correlation
Ventana benchmark xt, by Roche (2 mentions)
Cell condition 1 solution, by Roche (1 mentions)
Ez prep, by Roche (1 mentions)
Optiview dab detection kit, by Roche (1 mentions)
Bx53 microscope, by Olympus (1 mentions)
Chromomap dab detection kit, by Roche (1 mentions)
Benchmark xt, by Roche (1 mentions)
Mca341r, by Bio-Rad (1 mentions)
Iview dab detection kit, by Roche (1 mentions)
Benchmark xt system, by Roche (1 mentions)
Ultraview universal alkaline phosphatase red detection kit, by Roche (1 mentions)
5 protocols using haematoxylin
1
Immunohistochemical Analysis of GROα Expression
2
Immunohistochemistry of Immune Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, tissue samples were sectioned at a thickness of 5 μm. Deparaffinization, rehydration, endogenous peroxidase activity inhibition, and antigen retrieval were all performed on the Ventana Discovery Ultra IHC (Roche Diagnostics) automated stainer. Slides were then incubated with primary antibodies, followed by DISCOVERY HQ and DISCOVERY HQ-HRP system, visualized with ChromoMap DAB detection Kit (Ventana). The slides were then counterstained with haematoxylin (Ventana) and coverslipped. CD4 rabbit monoclonal 1:100, CD8a rabbit monoclonal 1:100, and CD11C rabbit monoclonal 1:100 were all purchased from Cell Signaling and F4/80 rat monoclonal 1:200 from Bio-Rad.
3
Immunohistochemical Profiling of Breast Carcinomas
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IHC investigation for various surrogate markers was performed to substantiate the results of gene expression analysis and to identify basal-like breast carcinomas. Markers investigated included the RTK EGFR, HER2 and IGF-1R, the hormone receptors ER and PR, and the basal-like marker CK5/6 32 . Whole FFPET sections cut at 3 µm were stained with a Ventana Benchmark XT autostainer (Ventana Medical Systems). Details of the antibodies and methods employed are given in Table 3 .
Slides were counterstained with haematoxylin (Ventana). System and isotype controls were included. IHC investigation for ER and PR and for HER2 was performed according to up-to-date histopathological guidelines and recommendations 28 , 29 (link).
Immunostaining for EGFR and IGF-1R was evaluated semiquantitatively with a scoring system similar to that established for HER2 (0: no membrane-specific staining, 1+: weak, incomplete cell membrane staining in <10% of cells, 2+: weak or moderate staining of the complete cell membrane in > 10% of cells, 3+: strong, complete membrane staining in > 10% of cells. Immunostaining for CK 5/6 was evaluated according to the criteria of Dabbs et al. 33 (link) (0: no staining, R: single cells stained, 1+: 5-30% of cells stained, 2+: >30-60% of cells stained, 3+: > 60% of cells stained). The scores 0 and R were considered negative, 1+ to 3+ positive.
Slides were counterstained with haematoxylin (Ventana). System and isotype controls were included. IHC investigation for ER and PR and for HER2 was performed according to up-to-date histopathological guidelines and recommendations 28 , 29 (link).
Immunostaining for EGFR and IGF-1R was evaluated semiquantitatively with a scoring system similar to that established for HER2 (0: no membrane-specific staining, 1+: weak, incomplete cell membrane staining in <10% of cells, 2+: weak or moderate staining of the complete cell membrane in > 10% of cells, 3+: strong, complete membrane staining in > 10% of cells. Immunostaining for CK 5/6 was evaluated according to the criteria of Dabbs et al. 33 (link) (0: no staining, R: single cells stained, 1+: 5-30% of cells stained, 2+: >30-60% of cells stained, 3+: > 60% of cells stained). The scores 0 and R were considered negative, 1+ to 3+ positive.
4
Quantification of CD68+ cells in tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formalin‐fixed tissues were dehydrated, embedded in paraffin and sectioned at 4 μm. Immunostaining was fully calibrated on a Benchmark XT staining module (Ventana Medical Systems, Oro Valley, AZ, USA). After dewaxing and rehydrating, sections were treated with 1:100 anti‐CD68 (ED‐1, Serotec MCA341R) for 40 minutes. Slides were then incubated at 60°C for 1 hour and processed using a fully automated protocol. Detection was performed using an iView DAB Detection Kit (760‐091; Ventana Medical Systems). Slides were counterstained with haematoxylin (Ventana Medical Systems). After the run on the automated stainer was completed, slides were dehydrated consecutively in 70% Eth, 95% Eth and 100% Eth for 10 seconds each. Before coverslipping, sections were cleared in xylene for 10 seconds and mounted with Entellan.
To quantify interstitial CD68+ staining, the number of positive cells was counted in 20 randomly selected non‐overlapping fields per animal (×100 magnification), and the mean value was presented. For glomerular CD68+ staining, the number of positive cells was counted in 25 glomeruli per animal (×400 magnification). All measurments were conducted in a blinded fashion.
To quantify interstitial CD68+ staining, the number of positive cells was counted in 20 randomly selected non‐overlapping fields per animal (×100 magnification), and the mean value was presented. For glomerular CD68+ staining, the number of positive cells was counted in 25 glomeruli per animal (×400 magnification). All measurments were conducted in a blinded fashion.
5
TYRP1 Immunohistochemistry in Skin and Lymph Node Metastases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry for TYRP1 protein was carried out in 118 paraffin-embedded skin and lymph node metastases using the G3E6 antibody raised against the C-terminal TYRP1 (1 : 100, Abcam, Cambridge, UK) and the ultraView Universal Alkaline Phosphatase Red Detection Kit (Ventana, Tucson, AZ, USA) on the BenchMark XT System (Ventana). The sections were counterstained with haematoxylin (Ventana). Immunostaining was blindly examined by two independent pathologists. A score from 0 to 8 was calculated by adding a score reflecting the proportion of positively stained cells (non=0; ≤1/100=1; 1/100 to 1/10=2; 1/10 to 1/3=3; 1/3 to 2/3=4; and ≥2/3=5) to a score reflecting the staining intensity (none=0; weak=1; intermediate=2; and strong=3).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!