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. Western blot analysis of extracts from OVKATE, DU 145, and MCF7 cells using VISTA ((D1L2G™) XP ® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using VISTA (D1L2G™) XP ® Rabbit mAb performed on the Leica® Bond™ Rx. Immunohistochemical analysis of paraffin-embedded human breast carcinoma using VISTA (D1L2G™) XP ® Rabbit mAb. Immunohistochemical analysis of paraffin-embedded human colon carcinoma using VISTA (D1L2G™) XP ® Rabbit mAb.

Immunohistochemical analysis of paraffin-embedded human non-Hodgkin's B-Cell lymphoma using VISTA (D1L2G™) XP ® Rabbit mAb. Immunohistochemical analysis of paraffin-embedded OVKATE (left) and MCF7 (right) cell pellets using VISTA (D1L2G™) XP ® Rabbit mAb. Immunohistochemical analysis of paraffin-embedded human kidney using VISTA (D1L2G™) XP ® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

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Immunohistochemical analysis of paraffin-embedded human tonsil using VISTA (D1L2G™) XP ® Rabbit mAb. Flow cytometric analysis of human whole blood cells stained with VISTA (D1L2G™) XP ® Rabbit mAb and co-stained with CD19-PE and CD14-APC.

VISTA is present on CD14+ monocytes (right; upper right quadrant) and shows a significant increase in intensity when compared to its isotype control (middle; upper left quadrant). CD19+ B cells are negative for VISTA (left; upper left quadrant). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor ® 488 Conjugate) #4412 was used as a secondary antibody.

Western Blotting Protocol For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween ® 20 at 4°C with gentle shaking, overnight. NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution. Solutions and Reagents From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: Western Blotting Application Solutions Kit NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water. 20X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH 2O, mix.

10X Tris Buffered Saline (TBS): To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH 2O, mix. 1X SDS Sample Buffer: Blue Loading Pack or Red Loading Pack Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH 2O. 10X Tris-Glycine SDS Running Buffer: To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH 2O, mix. 10X Tris-Glycine Transfer Buffer: To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH 2O, mix.

10X Tris Buffered Saline with Tween ® 20 (TBST): To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH 2O, mix. Nonfat Dry Milk:. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well. Wash Buffer: 1X TBST. Bovine Serum Albumin (BSA):. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well. Biotinylated Protein Ladder Detection Pack:.

Prestained Protein Marker, Broad Range (11-190 kDa):. Blotting Membrane and Paper: This protocol has been optimized for nitrocellulose membranes.

Pore size 0.2 µm is generally recommended. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody. Pc games mystery adventure.

Detection Reagent: SignalFire™ ECL Reagent. Protein Blotting A general protocol for sample preparation. Treat cells by adding fresh media containing regulator for desired time. Aspirate media from cultures; wash cells with 1X PBS; aspirate. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate).

Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity). Heat a 20 µl sample to 95–100°C for 5 min; cool on ice. Microcentrifuge for 5 min. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

NOTE: Loading of prestained molecular weight markers (, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (, 10 µl/lane) to determine molecular weights are recommended. Electrotransfer to nitrocellulose membrane. Membrane Blocking and Antibody Incubations NOTE: Volumes are for 10 cm x 10 cm (100 cm 2) of membrane; for different sized membranes, adjust volumes accordingly.

Membrane Blocking. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature. Wash three times for 5 min each with 15 ml of TBST. Primary Antibody Incubation. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product datasheet) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.

Wash three times for 5 min each with 15 ml of TBST. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody ( at 1:2000) and anti-biotin, HRP-linked Antibody ( at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.

Wash three times for 5 min each with 15 ml of TBST. Proceed with detection (Section D).

Detection of Proteins Directions for Use:. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST. Prepare 1X SignalFire™ ECL Reagent by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. For 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film. Avoid repeated exposure to skin.

Posted June 2005 revised November 2013 Western Blot Reprobing Protocol Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended.

Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment.

This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody. Solutions and Reagents NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water. Wash Buffer: Tris Buffered Saline with Tween ® 20 (TBST-10X).

Stripping Buffer: To prepare 100 ml, mix 6.25 ml of 1M Tris-HCl pH 6.8, 10 ml of 20% SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H 20. Make buffer fresh just prior to use. Protocol. After film exposure, wash membrane four times for 5 min each in TBST. Best results are obtained if the membrane is not allowed to dry.

Incubate membrane for 30 min at 50°C in stripping buffer (with slight agitation). Wash membrane six times for 5 min each in TBST.

(Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO ® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film. Wash membrane again four times for 5 min each in TBST. The membrane is now ready to reuse.

Start detection at the 'Membrane Blocking and Antibody Incubations' step in the Western Immunoblotting Protocol. Immunohistochemistry (Leica ® Bond ™) NOTE: Please see product datasheet or product webpage for appropriate antibody dilution.

Step Reagents Time/Temperature 1. Dewax Bond ™ Dewax Solution, 100% Alcohol, Bond ™ Wash Solution Pre-programmed Leica ®Bond ™ 2. Antigen Retrieval Bond ™ Epitope Retrieval ER2 Solution 20 min., 100°C 3. Peroxide Blocking Peroxide Block. 5 min. WASH Bond ™ Wash Solution 3x 0:00 min.

Protein Block CST NGS or Animal-Free Blocking Solution 20 min. Primary Antibody Dilute in Antibody Diluent 30 min. WASH Bond ™ Wash Solution 3x 2:00 min. Secondary Detection Novolink Polymer. 10 min. WASH Bond ™ Wash Solution/Deionized Water Custom (see below) 7a. Visualization Mixed DAB Refine.

0:00 min. Visualization Mixed DAB Refine. 10 min. WASH Deionized Water 3x 0:00 min. Counterstain Hematoxylin.

5 min. WASH Deionized Water 3x 0:00 min. WASH Bond ™ Wash Solution 3x 0:00 min.

WASH Deionized Water 3x 0:00 min. Dehydration (Offline): Incubate sections in 95% ethanol two times for 10 sec. Repeat in 100% ethanol, incubating sections two times for 10 sec each.

Repeat in xylene, incubating sections two times for 10 sec each. Mount sections with coverslips and mounting medium. Custom wash: Bond ™ Wash Solution 2:00 Bond ™ Wash Solution Dispenser Type: OPEN 0:00 Bond ™ Wash Solution 2:00 Bond ™ Wash Solution Dispenser Type: OPEN 0:00 Bond ™ Wash Solution 0:00 Deionized Water 0:00.Reagent included in Bond ™ Polymer Refine Detection Kit Catalog No: DS9800 LEICA ® is a registered trademark of Leica ®Microsystems IR GmbH. Bond ™ is a trademark of Leica ®Biosystems Melbourne Pty. No affiliation or sponsorship between CST and Leica ®Microsystems IR GmbH or Leica ®Biosystems Melbourne Pty. Immunohistochemistry (Paraffin) A.

Solutions and Reagents NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water. Ethanol, anhydrous denatured, histological grade (100% and 95%). Deionized water (dH 2O).

Hematoxylin (optional). Wash Buffer:. 1X Tris Buffered Saline with Tween ® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween ® 20 to 900 ml dH 20, mix. SignalStain ® Antibody Diluent. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain ® Citrate Unmasking Solution (10X) with 225 mL of dH 2O.

3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H 2O 2 to 90 ml dH 2O. Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum. 1X Animal-Free Blocking Solution: to 4 mL of dH 2O add 1 ml of Animal-Free Blocking Solution (5X). Detection System: SignalStain ® Boost IHC Detection Reagents (HRP, Rabbit ). Substrate: SignalStain ® DAB Substrate Kit. Hematoxylin: Hematoxylin.

Mounting Medium: SignalStain ® Mounting Medium. Deparaffinization/Rehydration NOTE: Do not allow slides to dry at any time during this procedure. Deparaffinize/hydrate sections:. Incubate sections in three washes of xylene for 5 min each. Incubate sections in two washes of 100% ethanol for 10 min each. Incubate sections in two washes of 95% ethanol for 10 min each. Wash sections two times in dH 2O for 5 min each.

Antigen Unmasking For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining.

Wash sections in dH 2O three times for 5 min each. Incubate sections in 3% hydrogen peroxide for 10 min. Wash sections in dH 2O two times for 5 min each. Wash sections in wash buffer for 5 min. Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature. Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain ® Antibody Diluent to each section. Incubate overnight at 4°C.

Equilibrate SignalStain ® Boost Detection Reagent (HRP, Rabbit ) to room temperature. Remove antibody solution and wash sections with wash buffer three times for 5 min each. Cover section with 1–3 drops SignalStain ® Boost Detection Reagent (HRP, Rabbit ) as needed. Incubate in a humidified chamber for 30 min at room temperature. Wash sections three times with wash buffer for 5 min each. Add 1 drop (30 µl) SignalStain ® DAB Chromogen Concentrate to 1 ml SignalStain ® DAB Diluent and mix well before use.

Apply 100–400 µl SignalStain ® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. Immerse slides in dH 2O. If desired, counterstain sections with hematoxylin. Wash sections in dH 2O two times for 5 min each.

Dehydrate sections:. Incubate sections in 95% ethanol two times for 10 sec each. Repeat in 100% ethanol, incubating sections two times for 10 sec each. Repeat in xylene, incubating sections two times for 10 sec each. Mount sections with coverslips and mounting medium. Flow Cytometry Alternative (Cell Surface) A.

Solutions and Reagents NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water. 20X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH 2O, mix.

16% Formaldehyde (methanol free). Triton™ X-100: To prepare 50 ml of 0.1% Triton™ X-100 add 50 μl Triton™ X-100 to 50 ml 1 X PBS and mix well. 50% methanol. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) in 100 ml 1X PBS. Store at 4°C. Secondary Antibodies:. B.

Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining. Aliquot 100 μl fresh whole blood per assay tube.

OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc. Add 65 μl of 10% formaldehyde to each tube. Vortex briefly and let stand for 15 min at room temperature. Add 1 ml of 0.1% Triton™ X-100 to each tube. Vortex and let stand for 30 min at room temperature.

Add 1 ml incubation buffer. Pellet cells by centrifugation and aspirate supernatant.

Repeat steps 7 and 8. Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use). Incubate at least 10 min on ice. Proceed with staining or store cells at -20°C in 50% methanol. Staining Using Unlabeled Primary and Conjugated Secondary Antibodies NOTE: Account for isotype-matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Add 2–3 ml incubation buffer to each tube and rinse by centrifugation.

Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer. Incubate for 30–60 min at room temperature. Wash by centrifugation in 2–3 ml incubation buffer. Resuspend cells in fluorochrome-conjugated secondary antibody diluted in incubation buffer according to the manufacturer’s recommendations.

Incubate for 30 min at room temperature. Wash by centrifugation in 2–3 ml incubation buffer. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer. Reference: Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV (2005) Cytometry A 67(1), 4–17. VISTA (V-Domain Ig Suppressor of T Cell Activation) is a negative checkpoint control protein that regulates T cell activation and immune responses. VISTA, which contains a single Ig-like V-type domain, a transmembrane domain, and an intracellular domain, has sequence similarity to both the B7 and CD28 family members.

Although primarily expressed by myeloid cells, VISTA is also expressed by CD4+, CD8+, and FoxP3+ T-cells. Thus, VISTA is described as both a ligand and a receptor (1-3). Blocking VISTA induces T-cell activation and proliferation, and potentiates disease severity in the EAE model (1). Furthermore, genetic deletion of VISTA in mice leads to spontaneous T-cell activation and chronic inflammation (4,5). In mouse models of cancer, neutralization of VISTA enhances T-cell proliferation and effector function and increases tumor infiltration, suggesting VISTA blockade could be an effective strategy for tumor immunotherapy (6,7). For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. BOND is a trademark of Leica Biosystems Melbourne Pty. No affiliation or sponsorship between CST and Leica Microsystems IR GmbH or Leica Biosystems Melbourne Pty.

Ltd is implied. LEICA is a registered trade​mark of Leica Microsystems IR GmbH. Tween is a registered trademark of ICI Americas, Inc.