Immu noh ist cemi ry F-la tgS s O com Reagents For Every Sense · Immu noh ist cemi ry F-la tgS s....
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Osenses.com Reagents For Every Sense v a Osenses Pty Ltd . 52 Torresan Crescent . Flagstaff Hill SA 5159 . Australia p a Po Box 1071 . Pasadena 5042 . Australia p.f 61-8-827 0 8586 . e [email protected] . w www.osenses.com Page 1 of 5 Starting Date: 1. Primary antibody to be tested Dilutions: 1:200 1:500 1:1000 1:2000 1:3000 1:4000 other: Diluent in which the antibody has been diluted: Complete name of the Tissue/Slide: Thickness of the tissue (μm): 5 10 15 20 30 35 40 other: Identification & number of the slides: Note: 2. Positive control Dilutions: 1:200 1:500 1:1000 1:2000 1:3000 1:4000 other: Diluent in which the antibody has been diluted: Complete name of the Tissue/Slide: Thickness of the tissue (μm): 5 10 15 20 30 35 40 other: Identification & number of the slides: Note: Immunohistochemistry on Free-Floating Sections
Transcript of Immu noh ist cemi ry F-la tgS s O com Reagents For Every Sense · Immu noh ist cemi ry F-la tgS s....
Osenses.com
Reagents For Every Sense
v a Osenses Pty Ltd . 52 Torresan Crescent . Flagstaff Hill SA 5159 . Australia p a Po Box 1071 . Pasadena 5042 . Australia
p.f 61-8-827 0 8586 . e [email protected] . w www.osenses.com Page 1 of 5
Starting Date: 1. Primary antibody to be tested Dilutions: 1:200 1:500 1:1000 1:2000 1:3000 1:4000 other: Diluent in which the antibody has been diluted: Complete name of the Tissue/Slide: Thickness of the tissue (µm): 5 10 15 20 30 35 40 other: Identification & number of the slides: Note: 2. Positive control Dilutions: 1:200 1:500 1:1000 1:2000 1:3000 1:4000 other: Diluent in which the antibody has been diluted: Complete name of the Tissue/Slide: Thickness of the tissue (µm): 5 10 15 20 30 35 40 other: Identification & number of the slides: Note:
Immunohistochemistry on Free-Floating Sections
Osenses.com
Reagents For Every Sense
v a Osenses Pty Ltd . 52 Torresan Crescent . Flagstaff Hill SA 5159 . Australia p a Po Box 1071 . Pasadena 5042 . Australia
p.f 61-8-827 0 8586 . e [email protected] . w www.osenses.com Page 2 of 5
3. Negative control Dilutions: 1:200 1:500 1:1000 1:2000 1:3000 1:4000 other: Diluent in which the antibody has been diluted: Complete name of the Tissue/Slide: Thickness of the tissue (µm): 5 10 15 20 30 35 40 other: Identification & number of the slides: Note: 4. Secondary antibody Name of the supplier: Dilutions: 1:200 1:500 1:1000 1:2000 1:3000 1:4000 other: Diluent in which the antibody has been diluted: Complete name of the Tissue/Slide: Thickness of the tissue (µm): 5 10 15 20 30 35 40 other: Identification & number of the slides: Note:
Osenses.com
Reagents For Every Sense
v a Osenses Pty Ltd . 52 Torresan Crescent . Flagstaff Hill SA 5159 . Australia p a Po Box 1071 . Pasadena 5042 . Australia
p.f 61-8-827 0 8586 . e [email protected] . w www.osenses.com Page 3 of 5
Protocol
• Day 1
Step 1: Elimination of endogenous peroxidase activity
Add 500 µl/well 1% H2O2, 50% Methanol in dH2O (15 ml 100% Methanol + 15 ml dH2O + 1 ml 30% H2O2). Place tissues in the appropriate wells. Incubate for (10 min @ RT with slow shaking).
Step 2: Wash Discard the 1% H2O2, 50% Methanol in dH2O using a pipette. Ensure the tissues remain in the wells. Wash the tissues with 1% NHS in 1xTBS-azide + 0.25% Triton X-100, 250-500 µl/well (1 x 10 min @ RT with slow shaking)
Step 3: Blocking non-specific binding sites Discard the 1% NHS-TBS-azide. Ensure the tissues remain in the wells. Block the non-specific binding sites with 20% NHS in 1xTBS-azide + 0.25% Triton X-100, 250-500 µl/well. Incubate for (1 hr @ RT with slow shaking).
Step 4: Primary antibody Discard the 20% NHS-TBS-azide. Ensure the tissues remain in the wells. Dilute the primary antibody in 1% NHS-TBS-azide + 0.25% Triton X-100. Add 250-500 µl/well (4-5 nights @ 4°C with slow shaking). Dilutions:
• Day 2
Step 5: Wash
Discard the Primary antibody. Wash tissues with 1% NHS - TBS-azide + 0.25% Triton X-100. Add ~250-500µl to each well (3 x 5 min @ RT with slow shaking).
Step 6: Secondary antibody (Biotinylated) Biotinylated donkey anti Rabbit/Sheep (Jackson) from Australian Laboratory Services.1:500 diluted in 1% NHS - TBS-azide + 0.25% Triton X-100, 250-500 µl/well (Overnight @ 4°C with slow shaking).
Osenses.com
Reagents For Every Sense
v a Osenses Pty Ltd . 52 Torresan Crescent . Flagstaff Hill SA 5159 . Australia p a Po Box 1071 . Pasadena 5042 . Australia
p.f 61-8-827 0 8586 . e [email protected] . w www.osenses.com Page 4 of 5
• Day 3 Step 7: Wash
Discard the secondary antibody. Wash tissues with 1x TBS. Add ~250-500µl to each well (3 x 5 min @ RT with slow shaking)
Step 8: ABC solution 1 drop reagent A + 1 drop reagent B in 5 ml 1x TBS (Prepare at least 30 mins before use and place on shaker) 250-500 µl/well (1 hr @ RT with slow shaking).
Step 9: Wash 1x TBS (3 x 5 min @ RT with slow shaking) Step 10: DAB reaction
1 tablet (Sigma) dissolved in 5ml 1x TBS + 3.5 µl 30% H2O2 , 250-500 µl/well (until colour develops ~ 5-10 min @ RT with slow shaking) Note: Dissolve tablet in 1ml dH2O, then top up to 5ml with 1x TBS
Step 11: Stop reaction Discard the DAB solution directly into 50% Bleach. Stop reaction with 1x TBS-azide.Place tissues on Superfrost slides. Allow to dry overnight (minium one night).
• Day 4 Step 12: Tissue Dehydration
Place slides in dH2O 5 min 70% Ethanol 5 min 95% Ethanol 5 min 100% Ethanol 1 5 min 100% Ethanol 2 5 min Xylene 1 10 min Xylene 2 10 min Cover slip Depex Allow drying overnight
Osenses.com
Reagents For Every Sense
v a Osenses Pty Ltd . 52 Torresan Crescent . Flagstaff Hill SA 5159 . Australia p a Po Box 1071 . Pasadena 5042 . Australia
p.f 61-8-827 0 8586 . e [email protected] . w www.osenses.com Page 5 of 5
RESULTS
Slide Dilution Specificity +ve Staining Intensity
Background Intensity
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