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Category Archives: Protocols
Hematoxylin Eosin Staining Protocol
Hematoxylin Eosin Staining Protocol
Hematoxylin is a basic dye that stains acidic residues, such as in DNA which is found in the nuclei of cells. Basophilic structures appear blue with hematoxylin. Eosin is an acidic dye that stains the cytoplasm of cells by binding to protein residues that are basic. Acidophilic substances appear pink with eosin.
1. Remove paraffin through dehydration using xylene for 10 minutes.
2. Rehydrate sections by processing through alcohol dilutions, from 100% to 30%, for 5 minutes each, similar to immunohistochemistry protocol, and then water for 5 minutes.
3. Stain in hematoxylin for 8 minutes.
4. Rinse in running water for 2 minutes.
5. Stain in eosin for 2 minutes.
6. Rinse in running water for 1 minute.
7. Dehydrate in 30%, 50%, 70%, 95% and 100% alcohol for 5 minutes each.
8. Mount onto slide.
Frozen Section Immunohistochemistry Protocol
Frozen Section Immunohistochemistry Protocol
1. Fix section in 4% acetone for 10 minutes.
- 4% paraformaldehyde can also be used
2. Let section stain at room temperature for 10 minutes to dry.
3. Pass through one change of xylene solution for 30 minutes.
4. Hydrate for 5 minutes each in one change each of 100%, 96%, 80%, 50% alcohol and water dilutions.
- this step, alcohol with water, deparaffinizes and rehydrates the sections
5. Rinse in PBS 1x for 5 minutes.
6. Treat with hydrogen peroxide for 10 minutes.
- this step, hydrogen peroxide, inhibits endogenous peroxidase activity
7. Rinse in PBS 1x for 5 minutes.
8. Incubate with protein block agent for 5 minutes and remove excess of protein block.
9. Incubate with primary antibody for 1 hour.
10. Rinse in PBS 1x for 5 minutes.
11. Incubate with secondary antibody for 10 minutes.
- secondary antibody is biotinylated and enzyme-streptavidin conjugates replace the complex of avidin-biotin peroxidase
12. Rinse in PBS 1x for 5 minutes.
13. Incubate with streptavidin-peroxidase for 10 minutes.
14. Rinse in PBS 1x for 5 minutes.
15. Incubate with chromogen for 10 minutes.
- chromogen solution application produce the different colorimetric products visible to the eye through the microscopy, highlighting protein positive cells and the proteins cellular location (e.g. cell membrane, cytoplasm, nuclear staining)
16. Rinse in PBS 1x for 5 minutes.
17. Stain in hematoxylin for 2 minutes or until section stained thoroughly.
18. Rinse in running water for 1 minute.
19. Mount slide.
20. Repeat entire protocol with positive controls (e.g. specimens known to express specific antigen) and negative controls (e.g. specimens known to NOT express the specific antigen)
- this is to make sure both that the reaction can occur and that the antibody is specific for the antigen and does not generally stain the whole tissue/tissues
Posted in Protocols
Tagged Frozen section immunohistochemistry, Immunohistochemistry frozen section protocol, Immunohistochemistry protocols, Immunohistochemistry technique
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Immunohistochemistry Protocol
Immunohistochemistry Protocol on Paraffin Sections
1. Pass through one change of xylene solution for 30 minutes.
2. Hydrate for 5 minutes each in one change each of 100%, 96%, 80%, 50% alcohol and water dilutions.
- this step, alcohol with water, deparaffinizes and rehydrates the sections
3. Rinse in PBS 1x for 5 minutes.
4. Treat with hydrogen peroxide for 10 minutes.
- this step, hydrogen peroxide, inhibits endogenous peroxidase activity
5. Rinse in PBS 1x for 5 minutes.
6. Incubate with protein block agent for 5 minutes.
7. Remove excess of protein block.
8. Incubate with primary antibody for 1 hour.
9. Rinse in PBS 1x for 5 minutes.
10. Incubate with secondary antibody for 10 minutes.
- secondary antibody is biotinylated and enzyme-streptavidin conjugates replace the complex of avidin-biotin peroxidase
11. Rinse in PBS 1x for 5 minutes.
12. Incubate with streptavidin-peroxidase for 10 minutes.
13. Rinse in PBS 1x for 5 minutes.
14. Incubate with chromogen for 10 minutes.
- chromogen solution application produce the different colorimetric products visible to the eye through the microscopy, highlighting protein positive cells and the proteins cellular location (e.g. cell membrane, cytoplasm, nuclear staining)
15. Rinse in PBS 1x for 5 minutes.
16. Stain in hematoxylin for 2 minutes or until section stained thoroughly.
17. Rinse in running water for 1 minute.
18. Mount slide.
19. Repeat entire protocol with positive controls (e.g. specimens known to express specific antigen) and negative controls (e.g. specimens known to NOT express the specific antigen)
- this is to make sure both that the reaction can occur and that the antibody is specific for the antigen and does not generally stain the whole tissue/tissues
Southern Blot
Southern Blot
Southern Blot Assay
Southern blot allows for the detection of DNA through binding of probes to DNA samples.
Microgram amounts of very high quality genomic DNA is used
Restriction enzymes are used to digest DNA into smaller fragments of various different sizes
DNA fragments are separated by size on an agarose gel by applying electrical charges through the gel by electrophoresis
DNA can be visualized by applying ethidium bromide and UV light to the gel containing the fragments
DNA is transferred to a membrane film, either made of nitrocellulose (most common) or nylon
The transfer to the membrane is done through blotting overnight.
The membrane is then hybridized to a labeled probe which is specific for a gene
Binding of the probe to the gene or specific fragment will highlight the presence of that genetic sequence within the sample utilized
Accurately detects large changes in DNA such as deletions and insertions
Requires large amounts of DNA that has to be of high quality
Southern blot is a long process that takes a couple of days to complete
Poor identification of point mutations and small deletions or insertions
Clinical Uses
Detecting deletions within genes
Shotgun Sequencing
Whole Genome Shotgun Sequencing Protocol
Genome Shotgun Sequencing Protocol Fleischman, 1995
1. Begin by random shearing of genome by ultrasonic waves
2. Clone genomic fragments into plasmid vectors
3. Transform these plasmid vectors into E.coli
4. Sequence the clones using fluorescent dideoxynucleotides
5. Align sequences with overlapping sequences
6. Identify open reading frames (ORFs)
7. Annotate the sequence
Plasmid Isolation
Plasmid Isolation Protocol
DNA Plasmid Isolation Protocol
This is the alkaline lysis protocol for DNA plasmid isolation.
1) E.coli are incubated in EDTA containg buffer which does not contain any divalent cations which can stabilize membranes and lysozymes which are needed to break down the bacterial wall. The EDTA chelates all divalent cations from the bacterial cells outer membrane.
2) Alkaline lysis solution is added containing SDS, which is an ionic detergent used to dissolve lipid membranes and NaOH, which has a high pH to denature double stranded DNA.
3) Quickly add ammonium acetate (NH4C2H3O2). Ammonium acetate acidifies the solution.
4) Pellet large chromosomal aggregates and any cellular debri from the dissolution of the bacteria through centrifugation.
5) Remove supernatant into a fresh tube which contains the small DNA, plasmids, and discard pellet, which conatins large DNA, chromosomes.
6) Ethanol is added to the supernatant to precipitate plasmid DNA, which is small enough not to get pelleted during centrifugation. Plasmid DNA renatures effectivately.
Electroporation
Electroporation
1. Pulsed electric fields to cells create small pores within the cell membrane
2. Once small pores are present within cell membrane, DNA placed along with the cells can enter through them
3. Must control pulse time, resistance and current
4. Can use both single stranded and double stranded DNA
Posted in Protocols
Tagged , dna, Electroporation, molecular biology, pores, protocol
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Bacteria Transformation
Bacteria Transformation
Gene Transformation
Gene transformation is a process of incorporated exogenous DNA into a recipient cell, with the final pathway being to cause genetic changes in the recipient cell.
Competence
Natural competence is the ability of a cell to efficiently take up DNA. Natural competence is physiological state which is genetically programmed.
Artificially Induced Competence
Artificially induced competence occurs when a cell can efficiently take up DNA after being treated with chemical or physical agents in order to be competent.
Calcium Induced Competence
Calcium induced competence is a form of artificially induced competence.
1. E.coli are treated with a high concentration of calcium ions
2. E.coli are then stored in the cold – this causes the E.coli cells to become transformable, but with a poor efficiency
3. Add double-stranded DNA, such as plasmids, along with reagents