Question: Can Mayer’s formulation instead of Gill II Hematoxylin be used for H&E Staining with Visium HD?
Answer: When performing the Visium HD assay, we recommend using Gill II Hematoxylin to achieve high quality H&E staining for this high resolution assay. Gill II formulation is more concentrated than Mayer’s, and therefore staining incubation times are shorter for the Gills II hematoxylin as compared to Mayer's. Also Bluing Buffer and Alcoholic Eosin incubation times have been optimized for use with Gills II hematoxylin and vary depending upon sample type (FF, FxF, FFPE) being used. The recommended Gill II Hematoxylin part numbers can be found in the Visium HD Spatial Gene Expression Protocol Planner (CG000698). We do not recommend using alternative Gill II Hematoxylin parts than those provided due to observing lower quality staining results.
Based upon experiments performed side-by-side with Mayer’s and Gill II formulation, staining quality was significantly improved with Gill II across all tissue types tested. Visium HD assay performance between the two dye formulations was comparable. Therefore, use of one of the Mayer’s Hematoxylin parts validated with the Visium CytAssist Spatial Gene Expression assay found in the Visium CytAssist Spatial Gene Expression H&E Staining Demonstrated Protocol (CG000520) should be low risk if the recommended Gill's II Hematoxylin parts can't be obtained.
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If utilizing Mayer’s Hematoxylin, replace the H&E staining step for Gill II with 1 ml Mayer’s hematoxylin and incubate for 3 minutes at room temperature while retaining the subsequent washing and staining steps in the H&E protocol.
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Steps to replace:
For FFPE - Chapter 3, steps 3.2 b & c of the Visium HD FFPE Tissue Preparation Handbook (CG000684).
For Fresh Frozen - Chapter 2, steps 2.4 b & c of the Visium HD Fresh Frozen Tissue Preparation Handbook (CG000763).
For Fixed Frozen - Chapter 2, steps 2.2 b & c of the Visium HD Fixed Frozen Tissue Preparation Handbook (CG00764).
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Steps to replace:
Zoomed in images from FFPE mouse brain hippocampus proximal sections demonstrating improved resolution of nuclear features utilizing Gill II Hematoxylin vs. Mayer's Hematoxylin.
Staining with Gill II Hematoxylin (Sigma, PN-GHS232)
Staining with Mayer's Hematoxylin (Sigma, PN-MHS16)
Product: Visium HD