Immunology

Chip Assay Protocol

Formaldehyde cross-linking and chromatin immunoprecipitation assays of
tissue culture cells are performed as described by Boyd and Farnham (MCB
29:8389-8399) and Y. Shang et. al. (Cell 103:843-852) with some modifications.
1. HC11 cells (2 x 107 in a 150 mm dish) are grown at confluence for three days
and primed for 24 hr in Priming Media [0.5 M glutamine/5 ??g/ml insulin/10%
stripped donor horse serum/RPMI 1640 media] in the presence of 1 ??g/ml
hydrocortisone.
2. Following the addition of 1 ??g/ml ovine prolactin for various times, cells are
cross-linked by adding formaldehyde to a final concentration of 1% (0.68 ml of
37%/25 ml media) directly into the media and rocked for 10 min at room
temperature.
3. Stop the cross-linking by adding glycine to a final concentration of 125 mM
(3.75 ml of 1 M/25 ml media). Continue to rock at room temp for 5 min.
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4. Wash the cell monolayers three times with ice-cold 1X PBS. Cells are then
scraped into 1X PBS (1 ml) plus protease inhibitors and collected by
centrifugation (700 x g for 4 min).
5. Cell pellets are resuspended in cell lysis buffer [5 mM Pipes (KOH), pH 8.0/85
mM KCl/0.5% NP-40] containing the following protease inhibitors 1 ??g/ml
leupeptin, 1 ??g/ml aprotinin and 1 mM PMSF and incubated for 10 min on ice.
The efficiency of cell lysis can be checked with trypan blue and if cells are not
lysed, they can be dounced on ice with a type B homogenizer several times.
6. Nuclei are pelleted by centrifugation (5000 rpm for 5 min).
7. Resuspend nuclei in nuclear lysis buffer [50 mM Tris, pH 8.1/10 mM
EDTA/1% SDS containing the same protease inhibitors as in cell lysis buffer].
Incubate on ice for 10 minutes.
8. Sonicate chromatin to an average length of about 600 bp while keeping
samples on ice. For HC11 cells, I sonicate on power setting 5 using a Branson
Sonifier 250 with a microtip in 20-sec bursts followed by 1 min of cooling on ice
for a total sonication time of 3 min per sample. This procedure results in DNA
fragment sizes of 0.3-1.5 kb.
9. Debris is cleared by centrifugation at maximum speed for 10 min at 4oC. At
this point, the supernatant containing the chromatin can be snap frozen in liquid
nitrogen and stored at -70oC for up to several months.
10. Transfer the supernatant to a new tube and dilute 5-fold in ChIP dilution
buffer [0.01% SDS/1.1% Triton X-100/1.2 mM EDTA, 16.7 mM Tris, pH 8.1/167
mM NaCl plus protease inhibitors].
11. To reduce nonspecific background, pre-clear the sample with 80 ??l of a
salmon sperm DNA/protein A agarose slurry for 30 min at 4oC with agitation.
12. Pellet beads by a brief centrifugation and collect supernatant fraction.
13. Save back 20% of the total supernatant as total input control and process
with the eluted Ips beginning with the cross-linking reversal step.
14. The rest of the supernatant is divided into two fractions: one for a no
antibody control and the second is incubated with 5 ??g of antibody overnight at
4oC with rotation.
15. Collect immune complexes with 60 ??l of the salmon sperm DNA/protein A
agarose slurry for 1 hr at 4oC with rotation.
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16. Beads were then washed consecutively for 3-5 minutes on a rotating
platform with 1 ml of each solution:
a. low salt wash buffer [0.1% SDS/1% Triton X-100/2 mM EDTA, 20
mM Tris, pH 8.1/150 mM NaCl]
b. high salt wash buffer [0.1% SDS/1% Triton X-100, 2 mM EDTA, 20
mM Tris, pH 8.1, 500 mM NaCl]
c. LiCl wash buffer [0.25 M LiCl/1% NP40/1% deoxycholate, 1 mM
EDTA/10 mM Tris, pH 8.0]
d. twice in 1X TE buffer
17. Elute complexes by adding 250 ??l of elution buffer [1% SDS/0.1 M NaHCO3]
to pelleted beads. Prepare buffer fresh each time. Vortex briefly to mix and
shake on vortexer for at least 15 minutes at setting “vortex 3”. Microfuge at
14,000 rpm for 3 minutes. Transfer supernatants to clean tubes. Repeat and
combine both elutions in the same tube.
18. Reverse formaldehyde crosslinks by adding 1 ??l 10 mg/ml RNase and 5 M
NaCl to a final concentration of 0.3 M to the elutants and incubate in a 65oC
water bath for 4-5 hours.
Add 2 1/2 volumes of 100% ethanol. Precipitate overnight at ???20oC.
19. Pellet DNA and debris and resuspend in 100 ??l of water. The input sample
will be gunky. Add 2 ??l of 0.5 M EDTA, 4 ??l 1 M Tris, pH 6.5 and 1 ??l of 20
mg/ml Proteinase K and incubate for 1-2 hours at 45oC.
20. Purify DNA using QiaQuick spin columns and elute in 50 ??l/column 10 mM
Tris, pH 8.0. Two ??l of DNA is used in quantitative PCR reactions.
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Supplemental Protocol: To make ssDNA/protein A agarose
(Courtesy of Upstate Biotechnology)
1. Make 100 ml of sterile TE (10 mM Tris, pH 8/1 mM EDTA, pH 8).
2. Combine: 50 mg BSA (the one used for diluting antibodies)
0.5 ml Sodium Azide (from a 5% stock solution)
Bring up to 47.5 ml with TE and filter sterize using a 0.2 ??
filter)
3. Wash 20 ml of protein A beads (50% slurry catalog 16-125, Upstate
Biotechnologies)
twice using 15 ml of sterile TE.
4. Combine: 10 ml washed protein A packed beads
4.0 mg salmon sperm DNA (sonicated)
bring up to 20 ml using sterile TE/BSA/sodium azide
solution
rock 45 minutes at 4oC.
5. Aliquot and store at 4oC.??

Double Immunofluorescence Staining For Bcl6 And Else

Any type of tissue is suitable for this technique, as long as the antigenicity for your antigen(s) is preserved.??
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Use cytocentrifuged cells or frozen or paraffin dewaxed sections.??
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Presence of endogenous fluorescent substances may affect your staining.??

Note:??This protocol uses a twice repeated sequence of primary and secondary layers in order to amplify the signal, as in the PAP/APAAP technique.

Start at this section for unfixed or acetone-fixed specimens.??

1.?? Mark the slide in order to recognize the area to be stained and label the slide.??
2.?? Fix in acetone for 10 min at RT [this step can be omitted].??
3.?? Blot the slides and let them dry for 10 min – 2 hrs.??
4.?? Fix in 10% formalin for 10 min in a Coplin Jar (From this point on, don’t let dry the slides at any time).??
5.?? Discard the formalin appropriately and quickly wash a few times in distilled water (from the distilled tap is OK).??

Start at this section for dewaxed, antigen retrieved slides??

6.?? Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added. 5 minutes/wash.??
7.?? Briefly blot the slides without letting them dry and then apply 3% human serum (health hazard!) or pig serum a s a blocking agent.
8.?? Incubate with the blocking for 10 min.??
9.?? Blot the slides without washing and apply a mixture of the primary antibodies, in a moist chamber, at RT for 1-18 hr.??
10. Wash twice.??
11. Apply a mixture of secondary antibodies (see example below) for 45 min at RT.??
12. Wash twice.??
13. Add the mixture of primary antibodies and incubate for 15 min.??
14. Wash twice .??
15. Add the mixture of secondary antibodies and incubate for 15 min.??

Proceed with steps 16 through 19 if one of the secondary is biotin-conjugated. Else go to 19.

16. Dilute the avidin-fluorochrome in TBS/Tween and centrifuge for 15 min.??
17. Wash thrice.??
18. Incubate with the avidin for 15 min.??
19. Wash thrice in TBS 0.05M pH7.5 + 0.01% Tween 20 and mount.

Example:

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Primary antibodies: rabbit anti-BCL-6 at 0.5??g/ml in PBS/BSA/Azide +??mouse anti HA supn 1:100.??
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Secondary antibodies: biotin-conjugated goat anti rabbit (1:200) + TRITC conjugated goat anti-mouse (1:200).?? Alternatively, biotin-conjugated goat anti mouse (1:200) + goat anti rabbit TRITC (1:200)

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Avidin-FITC: 1:300

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Double IF staining: BCL6 and Ki-67 (MIB 1)

Human tonsil, formalin fixed, paraffin embedded: Rabbit anti BCL-6 (TRITC, red) Mouse anti Ki-67 MIB 1 (FITC green) 40x.

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Human tonsil, squamous epithelium (formalin fixed, paraffin embedded section) 40x
?????????????????????????????????????? Mouse IgG2a anti Blimp-1 (anti mouse IgG2a, AMCA avidin),?? Mouse anti Ki-67 MIB 1 IgG1 (anti mouse IgG1-TRITC), Rabbit anti BCL-6 (anti rabbit FITC).

Immunohistochemistry Double Staining Protocol

Preface:

You should not co-stain in IHC antigens located on the same structure (nucleus, membrane, cytoplasm). That because the success of this double staining procedure is based on the complete development of the first stain, which should mask the structure stained first. Therefore you should be able to use two antibodies raised in the same species. However better results are obtained if two different species are used.

Careful check possible crossreactivty of your secondary antibodies by staining in simple IHC the relevant heterologous primary antibodies. E.g. many goat anti mouse IgG1??heavy chain do stain rat IgG2a??and IgG2b.

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Procedure:

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Apply the correct procedure (fixation or antigen retrieval) to the sections. Apply the blocking procedure. Be aware not to use for blocking any component which may be detected by any secondary antibody you plan to use.

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First Stain:??
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Indirect immunohistochemistry with avidin-biotin peroxidase(e.g rabbit anti antigen X, goat anti-rabbit biotin, avidin-HRP)

1.?? Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added.??
2.?? Briefly blot the slides without letting them dry and then apply 3% human or pig serum as a blocking agent (health hazard!).??
3.?? Incubate with the blocking for 10 min. If one of your antibody is biotin-conjugated, you need at this point to do endogenous biotin blocking.
4.?? Blot the slides without washing and apply the primary antibody, in a moist chamber, at RT for 1-18 hr.??
5.?? Wash twice in TBS 0.05M pH7.5 + 0.01% Tween 20.??
6.?? Add the biotin-conjugated secondary antibody (50 to 100 ??l) and incubate for 45 min. The secondary antibody should be absorbed against human serum; if not add 1% human serum before use.?? Most reagents are used at 1:100 or 1:200 dilution.??
7.?? Wash twice in TBS 0.05M pH7.5 + 0.01% Tween 20.??
7bis- block endogenous peroxidase by incubating in 0.1%NaN3??and 0.3% H2O2??for 30 min. Wash thrice.??
8.?? Add the HRP-conjugated avidin (50 to 100 ??l, dilution 1:300 -500 in nTBS-Tween) and incubate for 20 min. Be careful not to dilute the avidin in biotin-containing medium.??
9.?? Wash thrice in TBS 0.05M pH7.5 + 0.01% Tween 20.
10. Add 50 ml of?? the developing solution (see below ). Protect from direct light.??
11. After 5 min, check the staining in your positive and negative controls.??
12. Check the staining until complete, dense staining is obtained, but background is still low.??
13. When staining is complete, wash thoroughly in tap water.??
14. Transfer to TBS0.05M pH7.5 + 0.01% Tween 20.

HRP Developing Solution:

For 50 ml developing solution add in order:

  • Aminoethylcarbazole (20 mg tablets, Sigma A-6926, dissolved in 2.5 ml NN-DM formamide)

  • 50 ml acetate buffer pH 5.5 (52.5 ml of 0.1M acetic acid solution + 196.5 ml of a 0.1M Na acetate solution, bring to 500 ml)

  • 25 ??l H2O2??30%.

Shake well.
Filter with a 45??m filter (optional).??
Keep away from direct light, use within 5 min.

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Second Stain:??
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Double indirect immunohistochemistry (e.g rabbit anti antigen X, goat anti-rabbit AP, rabbit anti-goat AP)

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A second blocking step is omitted, because blocking is due to the first stain.??
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One can briefly boil the slides to re-retrieve antigens and quench the primary layer. (bring to a boil in EDTA buffer and let cool).

1.?? Apply the 2nd primary antibody, in a moist chamber, at RT for 1-18 hr.??
2.?? Wash twice in TBS 0.05M pH7.5 + 0.01% Tween 20.??
3.?? Add the AP conjugated secondary antibody (50 to 100 ??l) and incubate for 45 min. The secondary antibody should be absorbed against human serum; if not add 1% human serum before use.?? SBA Goat anti mouse AP or Goat anti Rabbit AP can be used 1:200 in TBS-BSA NaN3.??
4.?? Wash thrice in TBS 0.05M pH7.5 + 0.01% Tween 20.??
5.?? Add the AP conjugated tertiary antibody (50 to 100 ??l) and incubate for 15 min. The tertiary antibody should be absorbed against human serum; if not add 1% human serum before use.?? SBA Goat anti mouse AP or Goat anti Rabbit AP can be used 1:200 in TBS-BSA NaN3.??
6.?? Wash thrice in TBS 0.05M pH7.5 + 0.01% Tween 20.??
7.?? Add 50 ml of?? the developing solution (see below ). Protect from direct light.??
8.?? After 5 min, check the staining in your positive and negative controls.??
9.?? Check the staining at 10-15 min interval.??
10. When staining is complete (usually < 1 hr), wash thoroughly in tap water.??
11. Preferably postfix in formalin for 4-5 hrs before mounting in water soluble mounting medium (glycerol gelatin).

Do not counterstain, unless you can afford a very gentle hematoxilyn hue in the nuclei.

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AP Developing Solution:??
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For 50 ml developing solution add in order:

  • 50 ml Tris-Hcl 0.1M pH 9.2 (exact!) (1:10 from a stock solution 1M).

  • Levamisole 1mM (12 mg).
  • 20 mg Naphtol As BI phosphate (stock solution 40 mg/ml in NN-DM formamide, anhydrous, kept at -20??C).
  • 10 mg Fast Blue BB Diazonium salt (Sigma F3378).

Shake well.
Filter with a 45??m filter.??
Keep away from direct light, use within 5 min.

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Mouse spleen, 8 days post SRBC:??
BCL-6 nuclear stain (AEC, brown) and PNA cytoplasmic stain (Fast Blue BB, blue) 40x

Protocol For The Fluorescent Icc Staining Of Cell Smears

This protocol has been developed and optimized using R&D Systems??NorthernLights fluorescent secondary antibodies??but can be modified accordingly.

REAGENTS REQUIRED

  • Primary Antibodies
  • Blocking buffer: 10% normal donkey serum, 0.3% Triton?? X-100
  • DAPI (4′,6-diamidino-2-phenylindole) solution: Add 1 ??L of 14.3 mM stock for every 5 mL of PBS. Store any unused DAPI at 2-8 ??C, wrapped in aluminum foil
  • Deionized H2O
  • Dilution buffer: 1X PBS, 1% bovine serum albumin (BSA), 1% normal donkey serum, 0.3% Triton X-100, and 0.01% sodium azide
  • Anti-fade mounting medium
  • NorthernLights-conjugated secondary reagents??(or equivalent)
  • 1X PBS: 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4
  • Wash buffer: 0.1% BSA in 1X PBS

MATERIALS

  • Gelatin-coated microscope slide covered with monolayer cell smear.
  • Fine tweezers

PROCEDURE

  1. Wash the slides containing the fixed cells two times in 400 ??L of wash buffer
  2. Block non-specific staining by adding 400 ??L of blocking buffer, and incubate for 45 minutes at room temperature.
  3. Remove blocking buffer. No rinsing is necessary.
  4. Dilute the unconjugated primary antibody (or fluorescence-conjugated primary) in dilution buffer according to the manufacturer???s instructions. For fluorescent ICC staining of cell smears using R&D Systems antibodies, it is recommended to incubate at room temperature for 1 hour. Alternatively, incubate overnight at 2-8 ??C.
    Note:??Appropriate controls??are critical for the accurate interpretation of IHC/ICC results. All IHC/ICC experiments should include a negative control using the incubation buffer with no primary antibody to identify non-specific staining of the secondary reagents. Additional controls can be employed to support the specificity of staining generated by the primary antibody. These include absorption controls, isotype matched controls (for monoclonal primary antibodies), and tissue type controls.
  5. Wash two times in 400 ??L of wash buffer. If using a primary antibody with a direct fluorescent conjugate, go to step 8.
  6. Dilute the secondary antibody in dilution buffer according to the manufacturer???s instructions. Add 400 ??L to the wells, and incubate at room temperature for 1 hour in the dark. From this step forward, samples should be protected from light.
  7. Note: R&D Systems NorthernLights fluorescent secondary antibodies and streptavidin conjugates are bright, resistant to photobleaching, and are ideal for multi-color fluorescence microscopy.
    Note: If a biotinylated antibody was used in step 4, apply the appropriate NorthernLights Streptavidin conjugate in step 6.

  8. Rinse two times in 400 ??L of wash buffer.
  9. Add 300 ??L of the diluted DAPI solution to each well, and incubate 2-5 minutes at room temperature. DAPI binds to DNA and is a convenient nuclear counterstain. It has an absorption maximum at 358 nm and fluoresces blue at an emission maximum of 461 nm.
    Note: DAPI counterstain can obscure visualization of targets localized in cell nuclei.
  10. Rinse once with PBS and once with water.
  11. Carefully remove the coverslips from the wells and blot to remove any excess water. Dispense 1 drop of anti-fade mounting medium onto the microscope slide per coverslip.
  12. Visualize using a fluorescence microscope and filter sets appropriate for the label used. Slides can also be stored in a slide box at??<??-20 ??C for later examination.
    Note: Initial IHC/ICC studies often require further optimization and/or additional

Protocol For The Preparation And Fluorescent Icc Staining Of Nonadherent Cells

The following protocol has been developed and optimized by R&D Systems IHC/ICC laboratory for fluorescent ICC experiments using cell smears.

This protocol provides a basic guide for the preparation, fixation, and fluorescent staining of cell smear samples. Each investigator must determine the precise experimental conditions required to generate a strong and specific signal for each antigen of interest. If R&D Systems primary antibodies are employed, please refer to the product data sheets to obtain the recommended working dilutions. In the staining protocol, signal visualization is achieved using R&D Systems??NorthernLights?????range of fluorescent secondary antibodies and reagents. For all other reagents, please follow the manufacturer???s instructions.

Please read the protocol in its entirety before starting.

Protocol for the Preparation of a Cell Smear for Non-adherent Cell ICC

Smearing non-adherent cells across a gelatin-coated slide forms a monolayer of cells that can be easily visualized by ICC.

REAGENTS REQUIRED

  • Deionized H2O
  • Fixative: 4% formaldehyde in PBS (1x)
  • 1X PBS: 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4

MATERIALS

  • Microfuge tube (1.5 mL)
  • Gelatin-coated microscope slides
  • Hot plate
  • Hydrophobic barrier pen
  • Microfuge

PROCEDURE

  1. Fix the cells with 4% formaldehyde for 20 minutes at room temperature by adding formaldehyde directly to the culture media and adjust to approximately 1 x 106??cells/mL.
  2. Add 1 mL of the cell solution to a 1.5 mL microfuge tube.
  3. Spin down the cells for 30 seconds in a microfuge.
  4. Pour off the supernatant and resuspend the cell pellet in 1 mL of deionized H2O.
  5. Spin down the cells for 30 seconds in the microfuge.
  6. Pour off the supernatant, and resuspend the cell pellet in 200 ??L of deionized H2O.
  7. Add 5 ??L of the cell suspension to a gelatin-coated slide (3 spots per slide), and smear with the side of a pipette tip.
  8. Place the slide on a hot plate (low heat setting), and allow the liquid to evaporate.
  9. Check the slide under a microscope, and make sure that there are no salt crystals. If salt crystals are observed, wash the slide with water.
  10. Surround the cell spot with a hydrophobic barrier using a barrier pen and air dry.
  11. The slides can be stored at 2-8 ??C for up to 3 months or they may be stained immediately.

Nuclear Fast Red Counterstain Protocol

Nuclear Fast Red (Kernechtrot) Solution

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??????????????Nuclear fast red ————————- 0.1 g

???????????? Aluminum sulfat ———————— 5 g

??????????????Distilled water —————————100 ml

??????????????Dissolve aluminum sulfate (Sigma, Cat# 368458-500G) in water. Add nuclear fast red and slowly heat to boil and cool. Filter and add a grain of thymol as a preservative.

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Staining Pattern

Nuclear

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Suggested Use

Counterstaining??for immunohistochemistry or other special staining.

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Suggested Staining Time

5 minutes

Gills Hematoxylin Protocol

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Chemicals???? Single???? Double???? Triple Function????
Ethylene glycol???? 250 ml???? 250 ml???? 250 ml???? solvent????
Distilled water 750 ml 750 ml 750 ml???? solvent
Hematoxylin 2 g 4 g???? 6 g???? dye
Sodium iodate 0.2 g 0.4 g 0.6 g???? oxidizing agent
Aluminum sulfate???? 20 g???? 40 g???? 80 g???? mordant????
Glacial acetic acid 20 ml 20 ml???? 20 ml???? pH control

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Dissolve ethylene glycol in distilled water.

Add hematoxylin, then sodium iodate.

Add aluminum sulfate and acetic acid

Stir for 1 hour at room temperature.

Filter if necessary.

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Staining Characteristic

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Progressive

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Suggested Use

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Single strength for cytology.

Triple strength for immunohistochemistry??counterstain (dilute 1:5) for 30 seconds and H&E(dilute 1:5) for 5 minutes.

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Note

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This recipe gives clear and sharp nucleus staining with little background. Good for both immunohistochemistry counterstain and H&E. This solution is also alcohol and mercury free so it is good for IHC??counterstain??when AEC substrate??is used. When triple strength is used, dilute 1:5 with distilled water before use.

Abi1 Antibody Staining Protocol For Immunohistochemistry

Description:??ABI1 is a human homolog of mouse Abl-interactor-1 (Abi1), mapped on 10p11.2. ABI1 participate in the transduction of signals from Ras????to Rac by regulating Rac-specific guanine nucleotide exchange factor (GEF) activities. ABI1 dramatically promoted ABL1-mediated tyrosine phosphorylation of MENA, but not of other substrates. Abi-1 regulates c-Abl-mediated phosphorylation of Mena by interacting with both proteins. ABI1 plays a role in the leukemogenesis by translocating to MLL.

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Primary Antibody

Name:??ABI-1 IHC Antibody

Clone: Rabbit polyclonal

Supplier: IHC World

Catalog Number:??IW-PA1001

Dilution: Ready to Use

Incubation Time/Temp: 60 min/room temperature

Antigen Retrieval

Device: Incubator
Buffer/pH value:??IHC-TekTM??Proteinase K Solution (Cat# IW-1101)
Heat/Cool Temperature:??37 ??C/room temperature
Heat/Cool Time: 20 minutes/20 minutes

Detection Methods

Standard Method:??ABC Method??or??LSAB Method
Enhanced Method:??Polymeric Methods

Chromogen Substrate

Reagent: DAB
Incubation Time/Temperature: 1-3 minutes/room temperature

Counterstain

Reagent:??Mayer’s Hematoxylin
Staining Time: 30 seconds

Results:

Staining Pattern: Cytoplasmic
Images:??Search image


Additional Information:

Species Reactivity: Human, mouse, rat
Fixation: Formalin fixed paraffin embedded sections or acetone fixed frozen sections and cells.
Positive Control: Rat brain, spleen, human intestine, Hela cell.
Negative Control: Omit primary antibody, isotype control, absorption control
Blocking: Normal serum blocking is not needed for this RTU antibody; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary

8Ohdg Antibody Staining Protocol For Immunohistochemistry

Description:??8-OHdG (8-Hydroxy-2???-deoxyguanosine) is suitable for immunohistochemistry in formalin-fixed, paraffin embedded sections. 21 analogues of 8-OHdG were examined with only two exhibiting mini cross-reactivity. 8-sulfhydrylguanosine and 8-OHG demonstrate some cross-reactivity at high concentrations (at least 2 orders of magnitude were required for positive cross-reactivity.

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Primary Antibody

Name: Mouse Anti-8-OHdG Antibody

Clone: Mouse monoclonal

Supplier: Oxis International, Inc.

Catalog Number: 24326

Dilution: 1:20 using??IHC-TekTM??Antibody Diluent (Cat# IW-1000 or IW-1001)??to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60 min/room temperature

Antigen Retrieval

Device:??IHC-TekTM??Epitope Retrieval Steamer Set (Cat# IW-1102)
Buffer/pH value:??IHC-TekTM??Epitope Retrieval Solution (Cat# IW-1100)
Heat/Cool Temperature: 95-100????C/room temperature
Heat/Cool Time: 20 minutes/20 minutes

Detection Methods

Standard Method:??ABC Method??or??LSAB Method
Enhanced Method:??Polymeric Methods

Chromogen Substrate

Reagent: DAB
Incubation Time/Temperature: 1-3 minutes/room temperature

Counterstain

Reagent:??Mayer’s Hematoxylin
Staining Time: 30 seconds

Results:

Staining Pattern: Nuclear
Images:??Search image


Additional Information:

Species Reactivity: Human
Fixation: Formalin fixed paraffin sections
Positive Control: Skin
Negative Control: Omit primary antibody, isotype control, absorption control
Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary

Human Platelet Isolation Protocol

1. Approximately 8ml of human blood is collected in a vacutainer tube containing
acid citrate dextrose (yellow cap; 39 mM citric acid, 75 mM sodium citrate, and
135 mM glucose, pH 7.4).
2. Whole blood is centrifuged immediately at 190 x g for 15 min at room
temperature to obtain platelet-rich plasma (appr. 4ml; top fraction).
3. Count the platelets using a hemacytometer. Usual platelet counts range from
100-400 x10e3. The table below shows the number of platelets necessary for
each experiment.
4. To obtain a platelet pellet, centrifuge PRP at 2500 x g for 5 min at 22 ??C and
gently ressuspend the pellet in either PBS, Tyrode???s or other buffer w/o Ca2+.
Rest platelets at R.T for 10-15 min before use to prevent aggregation.
Ana M. D. Carneiro
Assay Number of Platelets Buffer
5-HT levels 100ul blood –
Uptake 1 x 10e7 Krebs-Ringer???s HEPES (KRH) buffer
containing 130mM NaCl, 1.3mM KCl,
2.2mM CaCl2, 1.2mM MgSO4, 1.2
mM KH2PO4, 1.8g/L glucose, 10mM
HEPES, 100??M pargyline, and
100??M ascorbic acid
SSRI Binding 1 x 10e8 100mM Tris-HCl, pH7.4; 100mM
NaCl
Immunoprecipitations 1 x 10e7 (100ug protein) Phosphate Buffered Saline
Western Blots 1 x 10e6 (10ug protein) Phosphate Buffered Saline