Standard Protocol for Immunohistochemistry

Materials Required
1. Slides
2. Phosphate buffered saline (pH 7.6)
3. Hydrogen peroxide
4. Primary antibody
5. Blocking serum (normal serum)
6. Biotinylated secondary antibody
7. ABC reagent (6, 7, and 8 are included in Vectastain Elite ABC kit)
8. Diaminobenzidine
9. Meyer's hematoxylin
10.Permount

Method
1. Incubate in a dry oven at 62oC for 1 hour. Slides should be maintained in a vertical orientation
to allow complete removal of the paraffin.
2. Dewax slides in xylene for 5 x 4 minutes.
3. Hydrate slides in 100%, 95%, and 75% ethanol for 2 x 3 minutes each.
4. Immerse slides in tap water for 5 minutes
5. Antigen retrieval method (optional).
6. Quenching of endogenous peroxidase (optional)
a.immerse slides in 3% hydrogen peroxide solution for 6 minutes.
b.wash slides in PBS for 3 x 5 minutes.
7. Incubate slides with blocking serum (1:50) for 30 min.*
8. Blot excess serum from section, and incubate with primary Ab. Suggested incubation time
(may vary between antibodies):
mAb 2 hours at room temperature or overnight at 4oC.
pAb: 1 ~ 1.5 hours at room temperature.
9. Wash slides in PBS for 3 x 5 minutes.
10.Incubate slides with biotin-conjugated secondary Ab for 30 min.*
11.Wash slides in PBS for 3 x 5 minutes.
12.Incubate slides with Avidin-Biotin Complexes for 30 min.*
13.Wash slides in PBS for 3 x 5 minutes.
14.Incubate slides in fresh DAB solution for 2 minutes. (We use DAB solution in Vector
DAB/Ni substrate kit).**
15.Stop the reaction by washing in tap water.
16.Counterstain in Meyer's hematoxylin for 10 seconds.
17.Dehydrates slides in 75%, 80%, 95% and 100% ethanol
18.Clear slides in xylene 4 X 5 minutes.
19.Mount cover slide with Permount.


* Blocking serum, secondary antibody and avidin-biotin-peroxidase complexes are included in most of the
immunostaining kit. Our lab uses the ABC kit from Vector Lab (Vectastain Elite ABC kit).
** We use DAB solution in Vector/DAB/Ni substrate kit (Vector Labs., Cat. SK-4100).

HOW IS MONOCLONAL ANTIBODY PRODUCED EXACTLY

Process by which large quantities of antibodies (targeted against a particular antigen X) can be produced.

A mouse is immunized by injection of an antigen X to stimulate the production of antibodies targeted against X. The antibody forming cells are isolated from the mouse's spleen.

Monoclonal antibodies are produced by fusing single antibody-forming cells to tumor cells grown in culture. The resulting cell is called a hybridoma.

Each hybridoma produces relatively large quantities of identical antibody molecules. By allowing the hybridoma to multiply in culture, it is possible to produce a population of cells, each of which produces identical antibody molecules. These antibodies are called "monoclonal antibodies" because they are produced by the identical offspring of a single, cloned antibody producing cell.

Once a monoclonal antibody is made, it can be used as a specific probe to track down and purify the specific protein that induced its formation

PLASMID EXTRACTION

Method
Many different methods are available for isolation of plasmid DNA. In this practical you will
be using a commercially available kit, which utilizes an affinity column to purify the plasmid
DNA. Bacteria (E. coli in this case) containing plasmid is grown from a single colony in a
suitable medium such as Luria Broth (LB). An aliquot of the culture is used for preparing
the plasmid DNA.

Centrifugation
Bacterial cult.Centrifugation results in pelleting of bacterial cells

Solution I

(Tris pH 8.0
EDTA- Ethylene diamine tetra acetic acid and
Glucose)
RNase
Lysozyme- optional

· Glucose gives osmotic shock that leads to the rupture
of cell wall and membrane
· EDTA, inhibits nucleases
· Rnase: Dergrades RNA
· Lysozyme is used for rupturing cell wall of bacteria.


Solution II
Sodium hydroxide
SDS- Sodium dodecyl sulphate
(Alkaline pH `12.0

Lyses the cell completely
· Alkaline pH denatures chromosomal DNA but not the
covalently closed circular plasmid DN

Solution III
Sodium or potassium acetate
(Acidic pH 5.4)

· Neutralizes the alkaline pH
· Precipitates protein and forms SDS-protein complex
· Chromosomal DNA renatures and aggregates with
protein

Centrifugation

· Pellets the Protein –DNA aggregates
· Plasmids will be present in the supernatant
(These plasmid can be precipitated with ethanol- This
used to be the conventional method)

Spin column
Silica membrane

· Silica membrane binds to plasmid DNA at high salt
buffer condition

Elution

· Under low salt condition (Distilled water), plasmids
can be eluted from the silica membrane

Protocol
Each group is provided with 10ml of an E.coli (containing plasmid) culture that has been grown
overnight at 37oC.
1. Transfer 1.5ml of bacterial culture to a micro centrifuge tube.
2. Pellet cells by centrifuging at 12,000 rpm for 2 minutes
3. CAREFULLY remove the supernatant.
4. Add another 1.5ml of culture to the same tube and centrifuge and repeat step 2 &3
5. Add 250 ml of Solution I /RNAase.
6. Resuspend the pellet by vortexing briefly or by pipetting up and down.
7. Add 250 ml of Solution II
8. Mix GENTLY by inverting and rotating the tube several times. DO NOT vortex!!!
9. Leave at room temperature for 5 minutes but NOT MORE than 5 minutes.
10. Add 350 ml of Solution III
11. Mix by inverting the tube 6-8 times
12. Centrifuge at 12,000 rpm for 12 min
13. While your tubes are spinning place a Spin column in a 2-ml collection tube.
14. After centrifugation, CAREFULLY transfer the supernatant to the column. DO NOT disturb the
pellet.
15. Centrifuge the column and collection tube at 12,000 rpm for 1 min
16. Discard the flow through collected in the collection tube.
17. Replace the column in the collection tube
18. Add 250 ml of HB Buffer into the column and centrifuge for 1 minute at 12,000 rpm and
discard the flow through.
19. Add 700 ml of wash buffer into the column and centrifuge for 1 minute at 12,000 rpm and
discard the flow through.
20. Centrifuge again to remove residual buffer for 90 seconds
21. Place the column in a sterile 1.5ml tube.
22. Add 50ml of deionised water or TE buffer into the column
23. Centrifuge at 12,000 rpm for 1 min.
24. Discard the column. Plasmid DNA is in the deionised water or TE buffer will be collected in the
1.5ml tube.
25. Place the tube containing plasmid DNA on ice for further use or store at –20oC

DNA ISOLATION FOR PLASMID

DNA ISOLATION FOR PLASMID ISOLATION

Reagents and solutions

1. Alkaline Solution I
50 mM glucose
25 mM Tris HCl (pH 8.0)
10 mM EDTA (pH 8.0)

2. 10 mg/ml lysozyme

3. Alkaline Solution II
0.2 N NaOH (2.0 ml of 10 N NaOH in 100ml double distilled water)
1.0% SDS

4. Alkaline Solution III
5 M Potassium acetate
Glacial acetic acid
Autoclaved water

5. TE buffer
10 mM Tris HCl (pH 8.0)
1.0 mM EDTA (pH 8.0)

6. Ring stand with clamp

7. 26 gauge needle

8. 1 cc syringe

9. Dialysis buffer
1 M Tris HCl (pH 7.5)
0.5 M EDTA (pH 8.0)
Autoclaved distilled water (1L)

10. Ampilcillin 100 µg/ml

11. Ice cold Isopropyl alcohol

12. Cesium chloride as per Plasmid DNA sample

13. Dialysis tubings (Nylon)

14. 5 M NaCl
29.22 g NaCl in 75 ml water and bring the volume to 100 ml.

15. Isoamyl alcohol

16. Luria Broth media
1 gm Tryptone
0.5 gm Yeast extract
1 gm Sodium chloride
Deionised water 75 ml
Shake all the solutes until dissolved. Adjust the pH to 7.0 with 5 N NaOH. Bring the volume of the solution to 100 ml with deionised water and sterilize it by autoclaving it for 20 minutes at 15 psi.

Procedure

Isolation of Plasmid DNA

1. Pick up a colony of bacteria and inoculate it in a conical flask containing 100 ml autoclaved Luria broth media supplemented with antibiotic (Ampicillin 100 µg/ml) and incubate overnight in a 37°C shaking water bath at 250 rpm.
2. Pour the culture in a 2.0 ml centrifuge tube and centrifuge at 5000 rpm for 20 minutes.
3. Discard the supernatant and wash the pellet in 1.0 ml double distilled water. Centrifuge the solution at 1000 rpm for 5 minutes.
4. Discard the supernatant and resuspend the pellet in 250µl cold alkaline solution I (stored at 4°C). Mix properly so that the pellet dissolves.
5. Add 20 mg of lysozyme in the above solution and mix well. Allow it to incubate on ice for 15 minutes.
6. Add 500 µl of alkaline solution II and mix properly. Mix the solution by flicking.
7. Now add 250 µl of alkaline solution III and incubate it on ice for 15 minutes.
8. Centrifuge the sample at 5000 rpm for 10 minutes and filter the supernatant into an autoclaved 2.0 ml centrifuge tube.
9. Fill the tube with the same amount of ice-cold isopropyl alcohol (stored at-20°C) and incubate in cold (-20°C) for 30 minutes.
10. Centrifuge the sample at 5000 rpm for 15 minutes. Discard the supernatant and air dry the pellet.
11. Resuspend the pellet in 500 µl TE buffer.

Purification of Plasmid DNA using Cesium Chloride

1. When the pellet is suspended completely, measure the mass of plasmid DNA by using a balance. For every gram of plasmid DNA solution, add 1.0 gm of solid cesium chloride. Warm the solution at 30°C to dissolve the CsCl salt.
2. Add 5 µl of 10mg/ml ethidium bromide to the above plasmid DNA solution.
3. Centrifuge the sample at 50,000 rpm for 10-15 hours at 20°C in an ultracentrifuge.
4. After the first run is complete remove the tube carefully and place it in the clamp and insert a needle just below the band. Remove the cap on the tube and pull out the band with the help of needle and syringe.
5. Place the sample in another ultra centrifuge tube and fill the tube with a solution of cesium chloride and TE. Spin at 50,000 rpm for 5 hours.
6. When the run is complete follow the above instructions for removing the bands.
7. Place the bands in a centrifuge tube and add equal volumes of TE and mix well. Now add isoamyl alcohol twice the volume of sample in the tube. Shake well and allow the phases to separate.
8. Pipet off the top pink layer and repeat the extraction with isoamyl alcohol until the top layer is clear and transparent.


Dialysis of Plasmid DNA

1. Prepare pieces of dialysis tubing by rinsing very well in autoclaved distilled water. Tie one end of the tube and carefully suspend he extracted plasmid DNA band into the tubing. Tie the other end too and place the sample in dialysis buffer.
2. Dialyze for at least 6 hours at 4°C using a magnetic stirrer. Repeat the process in new buffer solution.
3. Once the dialysis is complete, remove the liquid from the tubing and transfer it into another tube.
4. Add 1/10 volume of 5 M NaCl and twice the volume of pure ethanol.
5. Centrifuge the samples for 20 minutes at 5000 rpm at 4°C.
6. Discard the supernatant, leaving the pellet.
7. Rinse with 95% Ethanol and resuspend the pellet in 100 μl of TE buffer (pH 8.0) or autoclaved distilled water.

GENE IN INTERSEX

Previous genetic studies indicated intersex (ix) functions only in females and that it acts near the end of the sex determination hierarchy to control somatic sexual
differentiation in Drosophila melanogaster. We have cloned ix and characterized its function genetically, molecularly and biochemically. The ix pre-mRNA is not spliced, and ix mRNA is produced in both sexes. The ix gene encodes a 188 amino acid protein, which has a sequence similar to mammalian proteins thought to function as transcriptional activators, and a Caenorhabditis elegans protein that is thought to function as a transcription factor.

Bringing together the facts that (1) the ix phenotype is femalespecific and (2) functions at the end of the sex determination hierarchy, yet (3) is expressed sex nonspecifically and appears likely to encode a transcription factor with no known DNA-binding domain, leads to the inference that ix may require the female-specific protein product of the doublesex (dsx) gene in order to function. Consistent with this inference, we find that for all sexually
dimorphic cuticular structures examined, ix and dsx are dependent on each other to promote female differentiation. This dependent relationship also holds for the only known direct target of dsx, the Yolk protein (Yp) genes. Using yeast 2 -hybrid assay, immunoprecipitation of recombinant tagged IX and DSX proteins from Drosophila S2 cell extracts, and gel shifts with the tagged IX and DSXF proteins, we demonstrate that IX interacts with DSXF, but not DSXM. Taken together, the above findings strongly suggest that IX and DSXF function in a complex, in which
IX acts as a transcriptional co-factor for the DNA-binding DSXF

PRINCIPLE OF ELISA ......(HOW TO)....

Colleague from department medicine from Michigan has revealed about ELISA Technique in their chapter. The Following is their note :

1. Introduction
ELISA is the abbreviation of enzyme-linked immunosorbent assay. It is a useful and powerful method in estimating ng/ml to pg/ml ordered materials in the solution, such as serum, urine, sperm and culture supernatant (Savige, 1998). ELISA has been widely used in the life science researches (Ma, 1994; 2004).
The basic principle of an ELISA is to use an enzyme to detect the binding of antigen (Ag) antibody (Ab). The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab binding. An ELISA can be used to detect either the presence of Ags or Abs in a sample, depending on how the test is designed.

2. Materials and Methods
Homogenize tissue with 5 times of protein extract buffer  centrifuge 10,000 rpm 20 minutes  0.1 ml supernatant each well  over night at 4oC  PBS with 0.5% BSA washing 3 X 3 minutes  0.1 ml diluted primary antibody 1-2 hour at room temperature  PBS washing 3 X 3 minutes  0.1 ml diluted secondary antibody 1-2 hour at room temperature  PBS washing 3 X 3 minutes  dye (0.2 ml pNPP)  0.05 ml 3 N NaOH  O.D. (405 nm) measurement.

2.1 Extract buffer:
50 mM Tris-HCl or 50 mM HEPES (pH 7.4)
150 mM NaCl
0.02% sodium azide
0.1% SDS
0.1 mg/ml phenylmethylsulfonyl fluoride (PMSF)
0.001 mg/ml aprotinin
1% Nonidet P-40 (NP-40) or 1% Triton X-100
(The half-life of a 0.02 mM aqueous solution of PMSF is about 35 minutes. PMSF is usually stored as a 10 mM or 100 mM stack solution 1.74 or 17.4 mg/ml in isopropanol at –20oC).
2.2 Homogenize sample under ice.
2.3 Centrifuge sample at 10,000 rpm for 20 minutes at 4oC.
2.4 Keep supernatant at -70oC until usage.
2.5 PBS: Phosphate-buffered saline (PBS), pH 7.4, 1000 ml (NaCl 8 g, KCl 0.2 g, Na2HPO4 1.44 g, KH2PO4 0.24 g, adjust to pH 7.4 with HCl). Add 0.5% BSA of 1% milk into PBS when washing processed. It can also use Dulbecco's PBS or try others. Instead of BSA, it can use gelatin or milk. Skim (0.5% to 1%) milk could reduce the non-specific reaction.
2.6 Antibody: Primary and secondary antibodies are normally 1:1000 to 1:2000 diluted by PBS and 0.1 ml each well.
2.7 Dye: Use alkaline phosphatase yellow (pNPP) liquid substrate as the dye for the ELISA (Derango et al. 1996). This product is supplied as a ready-to-use buffered alkaline phosphatase substrate p-nitro-phenylphosphate (pNPP). Prior to reaction with alkaline phosphatase, the substrate should appear as a colorless to pale yellow solution. It will develop a yellow reaction product when reacted with phosphatase in microwell applications. For the end-point assays, the reaction can be stopped with 0.05 ml/well of 3 N NaOH for every 0.2 ml of substrate reaction. Following the reaction with alkaline phosphatase, a yellow reaction product forms can be read at 405 nm.

3. More Information
3.1 Using Polyclonal Antibodies:
(1) Antibody purification: Protein G column is the best for this purpose.
(2) Conjugate: Making conjugate is the most important part (e.g. horseradish peroxidase).
(3) 96-well plate: Making the solid phase using the 96-well plate.
3.2 Buffers and other reagents:
(1) Plate buffer: 0.1 M Sodium carbonate buffer, pH 9.5.
(2) Reaction buffer: 0.01 M Sodium phosphate buffer, pH 7.2, 0.15 M NaCl (PBS), 0.5% BSA, 0.05% thimerosal; You can also use Dulbecco's PBS or try others. Instead of BSA, you can use gelatin. Skim (0.5% to 1%) milk could reduce the non-specific reaction.
(3) Washing buffer: 0.05% Tween-20, 0.01 M Sodium phosphate buffer, pH 7.2 or 0.05% Tween-20, 0.15 M NaCl.
(4) Developing buffer: 0.05 M Sodium acetate buffer, pH 5.5.
(5) TMB stock solution: Tetramethylbenzidine 1 mg/ml in DMSO.
3.3 Making Conjugate:
(1) Nakane's method.
(2) Glutaraldehyde method.
(3) Maleimide method.
3.4 Steps:
(1) 2 mg Horseradish peroxidase (HRP) in 1 ml water: A.
(2) 21.4 mg NaIO4 (never to be NaIO3) in 1 ml water: B.
(3) 100 micro-l of B into A: Color will change to the dark green!
(4) Wait for 10 min at room temperature.
(5) Put into the dialysis tube (such as Molecular cut off 20,000).
(6) F. Put the tube into 5 mM NaAcetate buffer, pH 4.0 in a 2 to 3 l flask.
(7) Dialysis overnight: Color will change to the gold.
(8) Raise the pH of the HRP solution to pH = 9.0 by the addition of 0.2 M NaCarbonate buffer, pH 9.5 (try an aliquot of 0.05 ml).
(9) Mix with the antibody solution (8 mg of IgG in 1 ml), which has been pre-dialyzed to 0.01 M NaCarbonate buffer, pH 9.0 overnight.
(10) Incubate the mixture for 2 hr at room temperature.
(11) Put freshly prepared 0.1 ml, 0.1 M NaHBr4 in water to the solution.
(12) Incubate at 4 degree for 2 hr.
(13) Put the mixture into a dialysis tube and dialyze against PBS overnight.
(14) Now the conjugate solution is ready for use. Add thimerosal to a final concentration of 0.02% for preservation. Add glycerol to a final concentration of 10% (optional). If you stock the conjugate solution for a long period such as years, stock it at -80 degree. But, in this case, don't repeat freeze.
thaw. You can stock the solution at 4 degree at least 6 months.
3.5 Preparation of ELISA Plate: This will take 2 hr to overnight. Overnight is preferable.
(1) Dilute antibody (IgG) by Plate buffer: 5 to 10 micro-g/ml.
(2) Put the diluted antibody solution, 0.1 ml to the wells of 96-well ELISA plate.
(3) Incubate for 2 hr at room temperature or overnight at 4 degree.
(4) Discard the solution and wash the plate three times by washing buffer. Put 200 micro-l into wells using micro-pipette or just put the Washing buffer using some devices.
(5) Discard the Washing buffer by tapping against paper towel.
(6) Put 0.15 to 0.2 ml of reaction buffer. Now, the plate is ready for use. You can stock the plate at least for 6 months. Take care not to dry up the plate.
3.6 Using Monoclonal Antibodies:
(1) Antibody purification: Antibody purification step is the only special part comparing with materials and methods in using polyclonal antibody. For most monoclonals, except for IgM, Protein G column will be good for the practical use. If you failed by this method, confirm your procedure again before proceeding to the other methods such as DEAE column. When your monoclonal antibody is IgM, try Protamine column combined with molecular sieving column. Others are the same as above mentioned in "Using polyclonal antibodies.
(2) Try skim milk (any kind of powdered milk such as powdered milk for babies) instead of BSA: It's really cheap! Try 1% to 3%. It will decrease the background!! Thing is stability. It will form precipitate if you keep it for a few months. If you are running many plates, it is good alternative.

Correspondence to:
Hongbao Ma
138 Service Road, B410 Clinical Center
Michigan State University
East Lansing, MI 48824
Telephone: 517-432-0623; Email: hongbao@msu.edu

NEW TECHNOLOGY IN AUTISM DETECTION

Accoding to recent studies, a new type of genetic tests to detect abnormalities in children genes that lead to autism is said to be more accurate than standard tests that have been used before.

In this study, experts give three choices of tests on 933 people aged 13-22 years who had been diagnosed with autism. The three types of tests are, the G-banded karyotype test, chromosomal microarray analysis (CMA), and fragile X testing. Those three test is a type of test that is widely used.

Karyotype tests identify aberration chromosome associated with autism as much as 2 percent, while the fragile X genetic mutations found in 0.5 percent of patients. While CMA successfully detect chromosomal abnormalities more than 7 percent of the patients. Differences significant results was assessed to have greater levels of accuracy. Therefore, the experts suggested that CMA became the first test to determine the syndrome of autism in children.

The purpose of the genetic tests done on autistic children is to help parents determine will they have a child who is also autistic or not if their get pregnant again.

If the tests find abnormal chromosomes in the child, parents also need to do tests. If you found the abnormal gene, could be concluded that it is a high-risk that the parents will have children with autism again. However, if you find normal gene, there may be some duplication, so the risk of having autistic children is lower.

“In most cases, we believe there are at least a genetic predisposition to the occurrence of autism, but the ability of existing tests to identify specific genes is difficult because the technology does not exist. In addition, experts generally are not able to determine precisely the genetic mechanisms which trigger autism, “said Dr. Robert Marion, children geneticist from Montefiore Medical Center, New York, United States.

Today the practical standard to test whether a child suffers from autism are two types of genetic tests, karyotype and fragile X testing, which has been known since the 1960s.

Like karyotyping, CMA also sees an abnormal chromosome, but 100 times more accurate. CMA can recognize DNA duplication submicroscopic called re-number variants associated with autism.

Chromosome can be compared in a library and each book is his genes. “What we are looking for is a book which has a missing cover which describe the loss of an extra chromosome or chromosomal fragment containing the gene with autism,” said Dr. David Miller, a genetic expert.

Even so, there are still many things that can not be revealed by genetic experts. As many as 10-15 percent of cases of autism are not derived from a genetic disorder. Everything is still mysterious.