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.
A461 1
Manufactured by Thermo Fisher Scientific
The A461-1 is a laboratory centrifuge designed for general-purpose applications. It features a fixed-angle rotor capable of accommodating various sample tubes and microcentrifuge tubes. The centrifuge operates at adjustable speeds to facilitate various separation and sedimentation procedures.
Automatically generated - may contain errors
Lab products found in correlation
Cs702, by Agilent Technologies (3 mentions)
Ht110216, by Merck Group (3 mentions)
S3309, by Agilent Technologies (2 mentions)
F8775, by Merck Group (1 mentions)
Mayer s hematoxylin, by Agilent Technologies (1 mentions)
P7000, by Merck Group (1 mentions)
S6639, by Merck Group (1 mentions)
F8775 25ml, by Merck Group (1 mentions)
4 protocols using a461 1
1
Breast Cancer Tissue Staining and Imaging
2
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.
3
Methanol Fixation and H&E Staining for Visium Spatial Analysis
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The preparation of the mouse brain samples was performed following the Methanol Fixation, H&E Staining & Imaging for Visium Spatial Protocols (10x Genomics) [29 ]. In short, four sections at 8 μm of fresh frozen mouse brain (Adlego) were placed on a Visium Spatial Gene Expression slide and stored overnight at -80 °C. The slide was then transferred to 37 °C for 1 min before being immersed and fixated in methanol (VWR EU, 20847.307) at -20 °C for 30 min. After the fixation, the sections were dried by adding 500 μl isopropanol (Fisher Scientific, A461-1) for 1 min and then airdried. Next, the sections were stained with Mayer's Hematoxylin (Agilent, S23309) for 7 min, the slide was then washed in Milli-Q water, and then the sections were incubated in 1 ml of bluing buffer (Agilent, CS702) for 2 min. After another round of washing in Milli-Q water, 1 ml of Eosin mix (Sigma-Aldrich, HT110216, 1:10 dilution in Tris-Acetic Acid Buffer) was added and incubated for 1 min before washing. The tissue sections were then dried for 5 min at 37 °C and mounted with 85% glycerol (Merck, 104094) and a coverslip. Imaging was performed using the Metafer VSlide system at a magnification of 20x and the images were processed using the VSlide software. After imaging, the coverslip and remaining glycerol was washed off in Milli-Q water and the slide dried before transfer to the Slide Cassette.
4
Cross-linking Tissue for in situ RNA Analysis
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In experiments where cross-links were introduced a few key steps were performed differently. Fixation of tissue was done on the sRIN slide for 5 or 10 min at RT using PBS (09-9400, Medicago) containing 4% formaldehyde (F8775-25ML, Sigma-Aldrich), then washed briefly in PBS (09-9400, Medicago), dried for 1 min at 37 °C and then continued with propan-2-ol (A461-1, Fisher Scientific) treatment before HE staining. A tissue-specific (mouse olfactory bulb) permeabilization was performed before reverse transcription in situ. The permeabilization reactions were done, on the sRIN slide in a sealed hybridization cassette (AHC1X16, ArrayIT Corporation), first by adding a mixture of 1x Exonuclease I buffer (B0293S, NEB) and 0.2 mg/ml BSA (B9000S, NEB) for 30 min at 37 °C. Subsequently, tissue was briefly washed in 0.1 × SSC buffer (S6639, Sigma-Aldrich), treated with 0.1% pepsin (P7000, Sigma-Aldrich) dissolved in 0.1 M HCl (318965, Fluka) for 10 min at 37 °C, washed with 0.1 × SSC buffer (S6639, Sigma-Aldrich) and then reverse transcription in situ was performed immediately.
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