Apo-BrdU-IHC
Contents
1. BioVision rev 10 14 Apo BrdU IHC Jn Situ DNA Fragmentation Assay Kit Catalog K403 50 50 assays Store at 20 C INTRODUCTION Internucleosomal DNA fragmentation is a hallmark of apoptosis in mammalian cells BioVision s Apo BrdU IHC Kit is a two color TUNEL Terminal deoxynucleotide transferase dUTP Nick End Labeling assay for labeling DNA breaks to detect apoptotic cells by immunohistochemistry The kit contains positive negative control slides for assessing reagent performance reaction and blocking buffers for processing individual steps in the assay proteinase K terminal deoxynucleotidyl tranferase enzyme TdT bromodeoxyuridine triphosphate Br dUTP biotin labeled antiBrdU antibody for labeling DNA breaks horseradish peroxidase streptavidin conjugate DAB H2O2 Urea tablets for color generation and methyl green solution for counter staining the cells ll KIT CONTENTS Color Code Store Temp Part Number Control Slides pos neg natural box 2ea 20 C 403 50 1 Blocking Buffer white cap 22 ml 20 C 403 50 2 H O Urea Tablets amber vial 6ea 20 C 403 50 3 Protease K pink cap 0 11 ml 20 C 403 50 4 DAB Tablets amber vial 6ea 20 C 403 50 5 TdT Enzymes yellow cap 0 041 ml 20 C 403 50 6 Br dUTP violet cap 0 44 ml 20 C 403 50 7 200X Conjugate clear cap 0 035 ml 20 C 403 50 8 5X Reaction Buffer green cap 1 75 ml 4 C 403 50 9 Anti BrdU Biotin Antibody orange cap 0 275 ml 4 C 403 50 10 Methyl Green natural c2. Solution to complete the number of assays prepared per session The DNA Labeling Solution is active for approximately 24 hours 5X Reaction Buffer green cap TdT Enzyme yellow cap Br dUTP violet cap Distilled H20 Total Volume Carefully blot the 1X Reaction Buffer from the specimen taking care not to touch the specimen Immediately apply 50 ul of Complete Labeling Reaction Mixture prepared above onto each specimen except for the control slides which require only 25 pl each Note The use of a cover slip at this point assures even distribution of the reaction mixture and prevents evaporation during incubation Cover the specimen with a piece of Parafilm cut slightly larger than the specimen HINT Folding up one corner of the Parafilm cover slip will aid in its application and removal Place slides in a humid chamber and incubate at 37 C for 1 to 1 5 hours NOTE The DNA End Labeling Reaction can also be carried out at 22 24 C overnight for the control slides For samples other than the control slides provided in the kit incubation times at 37 C may need to be adjusted to longer or shorter periods depending on the characteristics of the tissue you used Remove Parafilm cover slip and rinse slide with PBS Gently tap off excess liquid and carefully dry the glass around the specimen Cover the entire specimen with 100 ul of Blocking Buffer white cap Incubate at room temperature for 10 minutes Carefully blot the Blocking Buf
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4. ap 6 ml room temp 403 50 11 lll GENERAL CONSIDERATIONS e The components of this kit are for Research Use Only To avoid reagent loss centrifuge vials before using e After initial defrosting the 5X Reaction Buffer and Anti BrdU Biotin Antibody should be stored at 4 C and Methyl Green should be stored at room temperature Do Not Refreeze e The control slides contain a mixture of apoptotic and non apoptotic cells allowing visualization of both positive amp negative labeling within the same microscope field e Incubation time for proteinase K DNase and the end labeling of the DNA may need to be empirically determined for your particular cell type and slide preparation Use this protocol as a starting guideline IV ASSAY PROTOCOLS Staining of Paraffin Embedded Tissue PET PET A Deparaffinization amp Rehydration 1 Immerse slides in xylene for 5 minutes at room temperature Repeat using fresh xylene for second 5 minutes incubation 2 Immerse slides in 100 ethanol for 5 minutes at room temperature Repeat using fresh 100 ethanol for second 5 minutes 3 Immerse slides in 90 ethanol for 3 min then 80 ethanol for 3 min and then 70 ethanol for 3 minutes at room temperature 4 Immerse slides briefly into 1X PBS and carefully dry the glass slide around the specimen If processing the kit s control slides simultaneously with unknown samples please refer to the CFS protocol CSF A At this point it may be helpful to encircl
5. around the specimen Cover the entire specimen with 100 ul of DAB solution Incubate at room temperature for 15 minutes Rinse slides with H2O and blot PET D Counterstain 22 22 23 24 Immediately cover the entire specimen with 100 ul of Methyl Green Counterstain natural cap solution Incubate at room temperature for 3 minutes Press edge of the slide against an absorbent towel to draw off most of the counterstain and place in a coplin jar slide holder Dip slides 2 times briefly into 100 ethanol Blot slides briefly on an absorbent towel Repeat step 22 using fresh 100 ethanol Blot slides briefly on an absorbent towel Dip slides into xylene or xylene substitute Wipe excess xylene from back of slide and around specimen 10 Mount a glass cover slip using a mounting media such as permount r over the specimen Mount a glass cover slip using a mounting media such as permount r over the specimen BioVision Inc 155 S Milpitas Blvd Milpitas CA 95035 USA Tel 408 493 1800 e Fax 408 493 1801 e Email tech biovision com e www biovision com BioVision sats V ASSAY PROTOCOLS Staining of Cell Preparations Fixed on Slides CFS The following protocol describes the method for measuring apoptosis in the positive and negative control slides that are provided in the APO BRDU IHC kit The same procedure should be employed for measuring apoptosis in your own slide specimens Important points to remember before starting t
6. e the specimen using a waxed pen or a hydrophobic marker Do not let tissue specimen dry out at any step If necessary cover or immerse specimen in 1X PBS to keep hydrated PET B Permeabilization Inactivation of Endogenous Peroxidase amp Equlibration 5 Dilute only enough Proteinase K pink cap needed 1 100 in 10 mM Tris pH 8 Cover the entire specimen with 100 ul proteinase K Incubate at room temperature for 20 minutes DO NOT OVER INCUBATE 6 Rinse slide with 1X PBS Gently tap off excess liquid and carefully dry the glass slide around the specimen 7 Dilute 30 H20 1 10 in methanol Cover the entire specimen with 100 ul of 3 H202 Incubate at room temperature for 5 minutes DO NOT OVER INCUBATE 8 Rinse slide with 1X PBS Gently tap off excess liquid and carefully dry the glass slide around the specimen 9 Dilute only enough 5X Reaction Buffer green cap as needed 1 5 with dH2O Cover the entire specimen with 100 ul of the 1X Reaction Buffer Incubate at room temperature for 10 to 30 minutes while preparing the labeling reaction mixture below BioVision Inc 155 S Milpitas Blvd Milpitas CA 95035 USA Tel 408 493 1800 e Fax 408 493 1801 e Email tech biovision com e www biovision com BioVision sais PET C End Labeling Reaction amp Detection 10 11 12 13 14 15 16 17 18 19 20 21 Prepare the Complete Labeling Reaction Mixture as follows Note Mix only enough DNA Labeling
7. eaker of 1X PBS Gently tap off excess liquid and carefully dry the glass slide around the specimen All the remaining steps of staining tissue cryosections on slides are identical to those steps outlined for staining of paraffin embedded tissue sections INACTIVATION OF ENDOGENOUS PEROXIDASES amp EQUILIBRATION PET B from Step 7 END LABELING amp DETECTION PET C COUNTERSTAIN PET D VI Technical Tips and Frequently Asked Questions About the APO BRDU IHC Assay 1 High background on slides all the cells are brown There are many possible explanations for this Did the sample dry out at any time during the staining BioVision Inc 155 S Milpitas Blvd Milpitas CA 95035 USA Tel 408 493 1800 e Fax 408 493 1801 e Email tech biovision com e www biovision com BioVision sated Did the H202 methanol solution evaporate off the sample in the inactivation of endogenous peroxidases step PET C Is this a false positive due to improper fixation of the tissue Was there a period of time between removal of the tissue and fixation when apoptosis or necrosis could have occurred Is their endogenous peroxidase activity Or non specific binding of strep avidin HRP Try staining a control with no TDT Some people suggest increasing the H20 concentration to 5 but over incubation with H202 or proteinase K can also damage DNA and create a false brown background 2 Cell fixation using a DNA crossing linking chemical fixative i
8. fer from the specimen taking care not to touch the specimen Immediately cover specimen with 100 ul of Antibody Solution prepared as described below Anti BrdU Biotin Antibody orange cap 5 ul 25 ul 50 ul Blocking Buffer white cap 95 ul 475 ul 950 ul Total Volume 100 pl 500 ul 1000 ul Incubate with the Antibody Solution in the dark for 1 1 5 hours at room temperature Hint Cover slides with aluminum foil Rinse slide in PBS Gently tap off excess liquid and carefully dry the glass around the specimen Cover the entire specimen with 100 ul of Blocking Buffer white cap Dilute only enough of the 200X Conjugate black cap needed 1 200 in Blocking Buffer white cap Prepared as described below 200X Conjugate black cap 0 5 ul 2 5 ul 5 0 ul Blocking Buffer white cap 100 ul 500 ul 1000 ul Carefully blot the Blocking Buffer from the specimen taking care not to touch the specimen Immediately apply 100 ul of diluted conjugate to the specimen Incubate at room temperature for 30 minutes Five minutes before concluding incubation prepare DAB solution by dissolving one tablet of DAB amber vial and one tablet of H2O2 Urea amber vial in one ml of tap H20 This yields enough DAB solution for 10 specimens Note Tap H2O may contain metal ions that enhance the DAB reaction DAB is highly carcinogenic and care should be taken when handling Rinse slides with 1X PBS Gently tap off excess liquid and carefully dry the glass slide
9. his assay The cells must be fixed prior to performing this assay DO NOT LET THE CELLS DRY OUT BETWEEN OR DURING ANY STEPS To avoid loss of cells from glass slides during washing steps it is recommended that slides be dipped into a beaker of 1X PBS rather than rinsed with a wash bottle CFS A Cell Fixation Rehydration amp Permeabilization 1 Pellet cells at 300xg for 5 minutes at 4 C Remove media Add enough 1 4 formaldehyde in PBS pH 7 4 to the pelleted cells to create a cell density of 1x10 cells ml and incubate 15 minutes at room temperature 2 Centrifuge at 300xg for 5 minutes at room temperature and resuspend at the same density in 70 ethanol An aliquot of fixed cells 100 300 ul can then be adhered to glass slides by directly placing the suspension onto the slide or by using a Cytospin Slides precoated with poly L lysine may enhance cell adherence 3 Immerse slides in 1X PBS for 10 minutes at room temperature Carefully dry the glass around the specimen At this point it may be helpful to encircle the specimen using a waxed pen or a hydrophobic slide marker 4 Dilute Proteinase K pink cap 1 100 in Tris pH 8 Cover the entire specimen with 50 100 ul of the diluted proteinase K Incubate at room temperature for 5 minutes DO NOT OVER INCUBATE 5 Dip slide 2 3 times into a beaker of 1X PBS Gently tap off excess liquid and carefully dry the glass slide around the specimen All the remaining steps of staining ce
10. lls fixed on slides are identical to those steps outlined in the previous section for staining of paraffin embedded tissue sections INACTIVATION OF ENDOGENOUS PEROXIDASES amp EQUILIBRATION PET B from Step 7 END LABELING amp DETECTION PET C COUNTERSTAIN PET D VI ASSAY PROTOCOLS Staining of Tissue Cryosections TCS Important points to remember before starting this assay Fixation of cryopreserved tissue is required prior to performing this assay DO NOT LET THE TISSUE DRY OUT BETWEEN OR DURING ANY STEPS if necessary cover or immerse the slide in 1X PBS to keep hydrated To avoid loss of tissue from glass slides during washing steps it is recommended that slides be dipped 2 3 times into a beaker of 1X PBS rather than rinsed with a wash bottle TCS A Tissue Fixation Rehydration amp Permeabilization 1 Immerse slides in 4 formaldehyde in PBS pH 7 4 for 15 minutes at room temperature Gently tap off excess liquid and carefully dry the glass slide around the specimen 2 Immerse slides in 1X PBS for 15 minutes at room temperature Carefully dry the glass slide around the specimen At this point it may be helpful to encircle the specimen using a waxed pen or hydrophobic slide marker 3 Dilute proteinase K pink cap 1 100 in 10 mM Tris pH 8 Cover the entire specimen with 50 100 ul of the diluted proteinase K solution Incubate at room temperature for 10 minutes DO NOT OVER INCUBATE 4 Dip slide 2 3 times into a b
11. s an important step in analyzing apoptosis Unfixed cells may lose smaller fragments of DNA that are not chemically fixed in place inside the cell during washing steps The researcher may have to explore alternative fixation and permeabilization methods to fully exploit their systems 3 Wash don t squirt your slides RELATED PRODUCTS Apoptosis Detection Kits amp Reagents e Annexin V Kits amp Bulk Reagents e Caspase Assay Kits amp Reagents Mitochondrial Apoptosis Kits amp Reagents Nuclear Apoptosis Kits amp Reagents Cell Fractionation System Mitochondria Cytosol Fractionation Kit Nuclear Cytosol Fractionation Kit Membrane Protein Extraction Kit Cytosol Particulate Rapid Separation Kit Mammalian Cell Extraction Kit e FractionPREP Fractionation System Cell Proliferation amp Senescence e Quick Cell Proliferation Assay Kit Senescence Detection Kit e High Throughput Apoptosis Cell Viability Assay Kits e LDH Cytotoxicity Assay Kit Cell Damage amp Repair HDAC Fluorometric amp Colorimetric Assays amp Drug Discovery Kits HAT Colorimetric Assay Kit amp Reagents DNA Damage Quantification Kit Glutathione Fluorometric amp Colorimetric Assay Kits GST Activity Assay Kit Nitric Oxide Fluorometric amp Colorimetric Assay Kits Signal Transduction e Camp amp cGMP Assay Kits e Akt amp JNK Activity Assay Kits e __ Beta Secretase Activity Assay Kit Adipocyte amp Lipid Transfer e Recombinant Adiponectin
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Apo BrdU IHC