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We have not tried counterstaining in-house, however, the paper below mentions using Hoechst as a fluorescent DNA dye to quantify the cells:
Baker, D. J. et al. (2004) BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice. Nat. Genetics 36, 744-749
You may be able to find other references on the web, as this kit is popular and there are many publications citing its use. We use hematoxylin routinely in our IHC experiments, and, consequently, have limited experience with alternatives.
For further suggestions, it may be helpful to seek out information within the Histonet (http://www.histosearch.com/histonet.html). This is a well-monitored discussion forum for histology professionals. There is an archive of queries that can be searched, or a new query can be posted. There may be suggestions or observations using these methods that could prove helpful.
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Our Cell Lysis Buffer #9803 will lyse whole cell membranes. Please keep in mind that sonication is crucial to maximize protein recovery when using Cell Lysis Buffer #9803 as sonication will ensure ample nuclear rupture and DNA shearing. Please refer to our recommended lysing protocol for Cell Lysis Buffer #9803 on the product webpage: http://www.cellsignal.com/products/9803.html. If you are looking to separate out fractions (cytoplasmic, membrane/organelle, nuclear/cytoskeletal) when lysing your cultured cells, feel free to consider our Cell Fractionation Kit (#9038).
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Our tissue extracts are prepared as follows:
- Cut a small piece of tissue (approximately 3 mm^3).
- Place in a clean Dounce homogenizer.
- Add 1mL cold 1X Cell Lysis Buffer #9803 + 1.0 mM PMSF #8553 + 1X Protease/Phosphatase Inhibitor Cocktail #5872.
- Homogenize well (approximately 20 strokes).
- Sonicate 3 times for 15 seconds each (cool on ice in between).
- Microcentrifuge at 12,000 rpm for 15-20 minutes to remove insoluble debris.
- Add 333uL of 3X SDS Sampler Buffer (adjust as needed depending on how much 1X Cell Lysis Buffer you added) or store at -80C in 1X Cell Lysis Buffer.
- Keep unused tissue on dry ice. …