Tissue sections from breast cancer specimens were first dried with isopropanol (Fisher Scientific, A461-1) before staining. The sections were then stained with Mayer’s hematoxylin (Agilent, S3309) for 4 min, washed in ultrapure water, incubated in bluing buffer (Agilent, CS702) for 2 min, washed in Milli-Q water and further incubated for 1 min in 1:20 eosin solution (Sigma-Aldrich, HT110216) in Tris-buffer (pH 6). The tissue sections were dried for 5 min at 37 °C and then mounted with 85% glycerol (Merck, 104094) and a coverslip. Imaging was performed using the Metafer VSlide system at ×20 magnification.
Ht110216
Manufactured by Merck Group
Sourced in United States
HT110216 is a laboratory equipment product manufactured by Merck Group. It is designed to perform specific functions in a laboratory setting. The core function of this product is to facilitate certain experimental or analytical processes, but a detailed description of its intended use is not available without the risk of making unsubstantiated claims.
Automatically generated - may contain errors
Lab products found in correlation
Cs702, by Agilent Technologies (4 mentions)
A461 1, by Thermo Fisher Scientific (3 mentions)
S3309, by Agilent Technologies (3 mentions)
Mhs16, by Merck Group (2 mentions)
Neo clear, by Merck Group (2 mentions)
Ht501128, by Merck Group (2 mentions)
Sp15 500, by Thermo Fisher Scientific (2 mentions)
Ds fi1, by Nikon (2 mentions)
Hematoxylin, by Merck Group (2 mentions)
Collagenase 1, by Thermo Fisher Scientific (1 mentions)
Pepsin, by Merck Group (1 mentions)
F8775, by Merck Group (1 mentions)
Dpx mounting medium, by Merck Group (1 mentions)
Ab175932, by Abcam (1 mentions)
Ab27591, by Abcam (1 mentions)
Sc5279, by Merck Group (1 mentions)
Ab15580, by Abcam (1 mentions)
Df505, by Oxford Instruments (1 mentions)
Dm4000b, by Leica (1 mentions)
Bp152 500, by Thermo Fisher Scientific (1 mentions)
18 protocols using ht110216
1
Breast Cancer Tissue Staining and Imaging
2
Tissue Histology and RNA Extraction
3
Spatial Transcriptomics Protocol for Tissue Sections
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The protocols used in our study have previously been described in Ståhl et al.24 (link),70 (link) and a detailed version of the entire protocol is available in Nature Protocols. In short, fresh frozen material was sectioned at 16 μm. After placing the tissue on top of the barcoded microarray, the glass slide was warmed at 37 °C for 1 min for tissue attachment and fixed in ~4% formaldehyde (Sigma-Aldrich, F8775) for 10 min at room temperature (RT). The slide was then washed briefly with 1× PBS (phosphate-buffered saline, Medicago, 09-9400). The tissue was dried with isopropanol (Fisher Scientific, A461-1) before staining. The tissue was stained with Mayer’s hematoxylin (Agilent, S3309) for 4 min, washed in Milli-Q water, incubated in bluing buffer (Agilent, CS702) for 2 min, washed in Milli-Q water, and further incubated for 1 min in 1:20 eosin solution (Sigma-Aldrich, HT110216) in Tris-buffer (pH 6). The tissue sections were dried for 5 min at 37 °C and then mounted with 85% glycerol (Merck, 104094) and a coverslip. Imaging was performed using the Metafer VSlide system at ×20 magnification. The images were processed with the VSlide software (v1.0.0). After the imaging was complete, the coverslip and remaining glycerol were removed by dipping the whole slide in Milli-Q water followed by a brief wash in 80% ethanol and warming for 1 min at 37 °C.
4
Paraffin Section Staining Protocol
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For paraffin sections, slides were incubated at 65 °C for 20 min to melt paraffin, followed by dewaxing in the first xylene solution (520860119, POCH) for 20 min and the second xylene solution for 5 min. Next, slides were rehydrated through incubation in a series of alcohols: absolute alcohol (396420113, POCH), 90% ethanol (EtOH) and 70% ethanol for 5 min each. After air drying, slides were stained with Mayer’s Haematoxylin solution (MHS16, Sigma–Aldrich) for 3 min in a Coplin jar, followed by rinsing in cool running water for 2 min. Then, the slides were stained with eosin water-based solution (HT110216, Sigma–Aldrich) for 1 min and dipped in running water until eosin stopped streaking. The slides were then dipped in 70% EtOH, 90% EtOH, absolute alcohol and xylene and finally mounted using DPX mounting medium (06522, Sigma–Aldrich). The next day, images were captured using an Axio Observer Systems Z1 microscope (Carl Zeiss Microscopy GmbH, Hannover, Germany) and analysed using Zeiss ZEN 2.5 lite Microscope Software (Carl Zeiss, Germany).
5
Histological Analysis of Estrous Cycle
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Fresh tissue samples were fixated for 24 h at 4 °C in 4% neutral buffered formaldehyde (stabilized with methanol), dehydrated, and embedded into paraffin. Subsequently, 3 µm thick sections were prepared, deparaffinized in xylene, and subjected to decreasingly graded ethanol for rehydration. Samples were stained with hematoxylin (#CS70030-2, DAKO, Agilent Technologies, Santa Clara, CA, USA) for 12 min and were afterwards placed under running tap water for a further 10 min. Slides were incubated in Eosin at a pH of 4.5 (#HT110216, Sigma-Aldrich) for 30 s and dehydrated in increasingly graded ethanol and xylene. DPX was used as a mounting medium (#06522, Sigma-Aldrich). The H & E stain was used for estrous cycle staging as previously described [49 (link),80 ,85 (link)]. Therefore, specific histological features of female reproductive tissues were used to distinguish between the different estrous stages, as summarized in Table S2 and Figure S5 .
6
Quantitative Thrombus Histology Analysis
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Mechanically analyzed thrombus samples were incubated in 4% paraformaldehyde at room temperature for 24 to 48 hours, after which they were embedded in paraffin and sectioned into 5 µm sections. Per sample, one section was stained with Hematoxylin and Eosin (H&E, HT110216, Sigma-Aldrich, St. Louis, MO) for the assessment of fibrin/platelets (on H&E, fibrin cannot be easily distinguished from platelets), erythrocytes and leukocytes. A second section per sample was stained with immunohistochemistry for platelets (CD61, CMC16121040, Cell Marque, Rocklin, CA). Microscopical images were acquired with a single slide scanner at 40× magnification (2.0 HT Nanozoomer, Hamamatsu, Japan), after which the quantitative fraction of histological components (relative to the total area of the section) was determined with the use of Orbit Image Analysis (version 3.15, Idorsia Pharmaceuticals Ltd, Allschwil).
7
Histological Evaluation of Murine Ankle Arthritis
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After completing the experiment, mice were sacrificed, and ankle samples were collected. Ankle specimens were fixed in 10% formalin for 1 week, decalcified in 10% formic acid at 37 °C for 1 week with shaking, and then embedded in paraffin. Paraffin blocks were sectioned at a thickness of 3.5 µm and deparaffinized using neo-clear (109,843, Merck, USA). Gradually graded ethanol was used for hydration, followed by staining with hematoxylin (105,174, Merck, USA) and eosin (HT110216, Sigma, USA). Additionally, safranin-O staining was performed on the joints to assess cartilage destruction. Two blinded readers independently scored the histological arthritis samples according to a previous report [14 (link)].
8
Histological Analysis of Embryonic Gonads
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For histology analyses, tissues were fixed in 4% paraformaldehyde (PFA), followed by dehydration, embedded in paraffin and processed into 5 μm sections for staining with haematoxylin (Sigma‐Aldrich, MHS16) and eosin (Sigma‐Aldrich, HT110216). Images were obtained with a Leica microscope (Leica, DM4000B). IHF was performed as previously described by Zhang et al.
27 (link) Briefly, embryonic gonads were fixed with 4% PFA in PBS at 4°C for 2 h and then embedded in paraffin. The 5 μm‐thick gonad sections were incubated with primary antibodies followed by washing three times with PBS containing 0.1% Triton X‐100. Nuclei were stained with DAPI after blotting with fluorochrome‐conjugated second antibodies. Images were obtained with a confocal microscope (Andor‐Oxford Instruments, DF505). Primary antibodies used in this study: OCT4 (used as 1:100 dilution, Santa Cruz, sc‐5279), STELLA (Sigma, 1:300, MAB4388); NRF1 (1:400, Abcam, ab175932), DDX4 (1:400, Abcam, ab27591), Ki67 (1:400, Abcam, ab15580) and cleaved Caspase‐3 (1:400, Cell Signalling Technology, #9661).
27 (link) Briefly, embryonic gonads were fixed with 4% PFA in PBS at 4°C for 2 h and then embedded in paraffin. The 5 μm‐thick gonad sections were incubated with primary antibodies followed by washing three times with PBS containing 0.1% Triton X‐100. Nuclei were stained with DAPI after blotting with fluorochrome‐conjugated second antibodies. Images were obtained with a confocal microscope (Andor‐Oxford Instruments, DF505). Primary antibodies used in this study: OCT4 (used as 1:100 dilution, Santa Cruz, sc‐5279), STELLA (Sigma, 1:300, MAB4388); NRF1 (1:400, Abcam, ab175932), DDX4 (1:400, Abcam, ab27591), Ki67 (1:400, Abcam, ab15580) and cleaved Caspase‐3 (1:400, Cell Signalling Technology, #9661).
9
Cryo-fixed Tissue H&E Staining Protocol
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The transported slides were placed on dry ice at –80°C and placed on a slide with tissue on a pre-warmed 37°C thermocycler adapter (10x genomics, 3000380). Then, the fixed tissues were fixed using ice-cold 100% methanol (Sigma, 34860) for 30 min and were stained with hematoxylin (Agilent, S330930-2) and eosin Y (Sigma, HT110216) (H&E) diluted 1:9 in 0.45M pH 6.0 Tris-buffer (Fisher, BP152-500) for 7 min and 1 min at room temperature, respectively. Between H&E staining, the glass slides were briefly dried, and bluing buffer (Agilent, CS70230-2) was added and washed off using RNase – and DNase – free Milli-Q water for 2 min. Then, we incubated the slide on the thermocycler adaptor with the thermal cycler (Thermo Fisher Scientific, 4375786) lid open for 5 min at 37°C and proceed to bright field imaging using a Leica SCN 400 slide scanner.
10
Histological Analysis of Embryonic Hearts
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Embryos were sacrificed by cervical dislocation and hearts were isolated and fixed with 4% paraformaldehyde overnight at 4 °C. After fixation the hearts were dehydrated in ethanol and stored at −20 °C for further paraffin embedding, sectioning and H&E staining. For histological analysis hearts were incubated in xylol and embedded in paraffin. Hematoxylin and eosin staining was performed according to the manufacturer’s instruction (Sigma, GHS116, HT-110216). Representative images of histological analysis of minimum six embryos with the same genotype are presented.
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