ISH Detection Kit (POD)

ISH Detection Kit Cited in 6 publication(s).

Product Info Summary

SKUMK1030
Pack size 1 kit (sufficient for staining100 slides)
ApplicationsISH

Overview

Product Name ISH Detection Kit (POD)
KU/Catalog Number MK1030
Size 1 kit (sufficient for staining100 slides)
Storage Store the kit at 4°C upon receipt. It is stable for one year at 4°C.
Applications ISH

Introduction

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or slide of tissue (in situ), or, if the tissue is small enough (e.g., plant seeds, Drosophila embryos), in the entire tissue (whole mount ISH), in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry, which usually localizes proteins in tissue slides. Boster’s ISH Detection Kit is highly sensitive, specific, fast and easy to perform. MK1030 is suitable for oligonucleotide probes conjugated by digoxin intensively. It is not recommended to be used for any oligonucleotide probe conjugated by only one digoxin.
Note: cDNA probe should be denatured prior to hybridization.

Kit Components

DescriptionQuantityVolumeCatalog Number
Pepsin (10X)12mLMK1030-1
Prehybridization Solution12mLMK1030-2
Oligonucleotide Probe Hybridization Diluent Solution12mLMK1030-3
Blocking Reagent15mLMK1030-4
Biotinylated Mouse Anti-Digoxin15mLMK1030-5
SABC-POD15mLMK1030-6
Biotinylated Peroxidase15mLMK1030-7

Material Required But Not Provided

1. Coverslips specialized for in situ hybridization.

2. POLY-L-LYSINE.

3. DEPC.

4. 20% glycerin buffer (3% citric acid, 2X sodium saline citrate, 0.5X sodium saline citrate, 0.2X sodium saline citrate, 0.5M PBS).

5. DAB Chromogenic Substrate Kit (Catalog Number: AR1022).

6. Hematoxylin Counterstain Solution (Catalog Number: AR0005).

Assay Procedure

ICC and IHC-F

Preparation of working buffer

3% citric acid: Add 3g citric acid into 100mL distilled water at a pH of around 2.0.

2X SSC: Add 17.6g NaCl and 8.8g Na3C6H5O7•2H2O into 1000mL distilled water.

0.5X SSC: Add 100mL 2X SSC into 300mL distilled water.

0.2X SSC: Add 30mL 2X SSC into 270mL distilled water.

20% glycerin: Add 20mL glycerin into 80mL distilled water.

0.5M PBS: Add 30g NaCl, 6g of Na2HPO4 • 12H2O, and 0.4g NaH2PO4 • 2H2O into 1000mL distilled water. Adjust pH to 7.2-7.6.


Note: Fix in time and add 0.1% DEPC into the stationary liquid to aviod that RNase decomposes mRNA.

1. 1. For ICC, Coverslip: treat with POLY-L-LYSINE or APES. Thickness of slide: 10-20μ m.
(For IHC-F, Microscope slide: treat with POLY-L-LYSINE or APES. Thickness of slide: 10-20μ m.)

2. Culture the cell on coverslip treated with POLY-L-LYSINE in the condition of 37°C and 5% CO2. Use Dulbecco as the basic culture media. Wash the grown cell with 0.1M PBS (pH 7.4) three times for 2 minutes.
(Note: Place a blank coverslip in a plate well. Add the culture media containing cells into the plate well and cells will grow naturally on the coverslip.)

3. Fix the slide with 4% Paraformaldehyde/0.1M PBS containing 1/1000 DEPC at room temperature for 20-30 minutes. Wash the slide with distilled water. It can store at -20°C for 2 weeks after the slide is dry.

4. Mix 30% H2O2 with CH3OH in the ratio of 1: 50. Deal the slide with the mixture at room temperature for 30 minutes to inactivate Endogenousperoxidase and wash with distilled water.

5. Exposure the fragments of mRNA nucleic acid. Pipette Pepsin that is diluted by 3% citric acid (Mix completely 1mL 30% citric acid with two drops of concentrated Pepsin) and digest at 37°C or room temperature for 5-120 seconds. Wash with 0.5M PBS 3 times for 5 minutes.

6. Prehybridization: Preparation for wet box—Add 20mL of 20% glycerin to the bottom of dry hybridization chamber to keep moist. Apply 20 µl of prehybridization solution per slide. Incubate at 37-40°C incubator for 2-4 hours. Absorb excess liquid and do not wash.

7. Hybridization: dilute digoxin-labeled oligonucleotide probe with hybridization diluent solution at a concentration of typically 0.5-2 μg/ml. Apply 20 µl of the mixing solution per slide. Remove the protective film of the coverslip specialized for in situ hybridization and place it on the slide. Hybridize overnight at 37-40°C incubator.

8. Wash: remove the coverslip and wash twice with 30-47°C 2X SSC for 5 minutes; wash once with 0.5X SSC for 15 minutes; wash once with 0.2X SSC for 15 minutes (Repeat as necessary).

9. Blocking: add blocking solution and blocking at 37°C for 30 minutes. Discard excess liquid and do not wash.

10. Add Biotinylated Mouse Anti-Digoxin: at 37°C for 1 hour or room temperature for 120 minutes. Wash 4 times with 0.5M PBS for 5 minutes. Do not wash with other buffers and distilled water.

11. Add SABC: at 37°C for 20 minutes or room temperature for 30 minutes. Wash 3 times with 0.5M PBS for 5 minutes. Do not wash with other buffers and distilled water.

12. Add Biotinylated Peroxidase: at 37°C for 20 minutes or room temperature for 30 minutes. Wash 4 times with 0.5M PBS for 5 minutes.

13. Color development: use DAB Chromogenic Substrate Kit (Catalog Number: AR1022) —add one drop (50μL) Reagent A (DAB Chromogen Concentrate), one drop (50μL) Reagent B (H2O2 Concentrate) and one drop (50μL) Reagent C (TBS buffer Concentrate) to 1mL distilled water and mix well. Then add the mixture to the specimen. Control color development under a microscope within 30 minutes. Continue to develop if there is no staining background. Wash the slide well with water.

14. Counterstain with Mayor’s hematoxylin if desired. Wash the slide well with water.

15. Dehydrate, clean in xylene and mount slide.


IHC-P

Harvest fresh tissue and fix in time with 4% Paraformaldehyde/0.1M PBS containing 1/1000 DEPC. Cut the larger specimen into slices of thickness of 4mm or less and fix for 1 hour. Large specimens should be fixed for less than 2 hours. Some tissues are particularly sensitive to excessive fixation, such as animal brains, which are fixed for 30-40 minutes and must not exceed 1 hour.

1. Dehydrate, wax and embed. Thickness of slide: 6-8μ m.

2. Microscope slide: treat with POLY-L-LYSINE or APES.

3. Immerse dewaxed paraffin slide into the 3% H2O2 at room temperature for 10 min. Wash the slide twice with distilled water.

4. Exposure the fragments of mRNA nucleic acid. Pipette Pepsin that is diluted by 3% citric acid (Mix completely 1ml 30% citric acid with two drops of concentrated Pepsin) and digest at 37°C or room temperature for 3-30 minutes (Digestion time depends on the thickness and freshness of specimens. Sufficient digestion enables mRNA exposure which enhance hybridization signal. However, overdigestion might decrease the thickness of the slide.). Wash with 0.5M PBS 3 times for 5 minutes. Wash once with distilled water.

5. Prehybridization: Preparation for wet box—Add 20mL of 20% glycerin to the bottom of dry hybridization chamber to keep moist. Apply 20 µl of prehybridization solution per slide. Incubate at 37-40°C incubator for 2-4 hours. Absorb excess liquid and do not wash.

6. Hybridization: dilute digoxin-labeled oligonucleotide probe with hybridization diluent solution at a concentration of typically 0.5-2 μg/ml. Apply 20 µl of mixing solution per slide. Remove the protective film of the coverslip specialized for in situ hybridization and place it on the slide. Hybridize overnight at 37-40°C incubator.

7. Wash: remove the coverslip and wash twice with 30-47°C 2X SSC for 5 minutes; wash once with 0.5X SSC for 15 minutes; wash once with 0.2X SSC for 15 minutes (Repeat as necessary).

8. Blocking: add blocking solution and blocking at 37°C for 30 minutes. Discard excess liquid and do not wash.

9. Add Biotinylated Mouse Anti-Digoxin: at 37°C for 1 hour or room temperature for 120 minutes. Wash 4 times with 0.5M PBS for 5 minutes. Do not wash with other buffers and distilled water.

10. Add SABC: at 37°C for 20 minutes or room temperature for 30 minutes. Wash 3 times with 0.5M PBS for 5 minutes. Do not wash with other buffers and distilled water.

11. Add Biotinylated Peroxidase: at 37°C for 20 minutes or room temperature for 30 minutes. Wash 4 times with 0.5M PBS for 5 minutes.

12. Color development: use DAB Chromogenic Substrate Kit (Catalog Number: AR1022) —add one drop (50μL) Reagent A (DAB Chromogen Concentrate), one drop (50μL) Reagent B (H2O2 Concentrate) and one drop (50μL) Reagent C (TBS buffer Concentrate) to 1mL distilled water and mix well. Then add the mixture to the specimen. Control color development under a microscope within 30 minutes. Continue to develop if there is no staining background. Wash the slide well with water.

13. Counterstain with Mayor’s hematoxylin if desired. Wash the slide well with water.

14. Dehydrate, clean in xylene and mount slide.

Result

The cytoplasm staining of positive cells is brownish yellow.

Note: Staining on a small amount of nuclear is normal. If staining on a large number of nuclear, it means that the fixation time is too longer and the specimen should be reprocessed. When there is other obvious non-specific staining, dilute the hybridization solution containing the probe with prehybridization solution at 2-5 times.

Product Images

MK1030 has been cited in 6 publications:

*The publications in this section are manually curated by our staff scientists. They may differ from Bioz's machine gathered results. Both are accurate. If you find a publication citing this product but is missing from this list, please let us know we will issue you a thank-you coupon.

Zeng Z,Teng Q,Xiao J.Long noncoding RNA ILF3-AS1 aggravates papillary thyroid carcinoma progression via regulating the miR-4306/PLAGL2 axis.Cancer Cell Int.2021 Jun 27;21(1):322.doi:10.1186/s12935-021-01950-8.PMID:34176471;PMCID:PMC8237480.
Species: Human,Mouse
MK1030 usage in article: APP:ISH, SAMPLE:THYROID TISSUE, DILUTION:NA

MicroRNA%u201134a suppresses colorectal cancer metastasis by regulating Notch signaling

Up-regulated miR-199a-5p in gastric cancer functions as an oncogene and targets klotho

Long non-coding RNA CTA sensitizes osteosarcoma cells to doxorubicin through inhibition of autophagy

MicroRNA%u2010224 inhibits progression of human prostate cancer by downregulating TRIB1

MicroRNA-148a inhibits breast cancer migration and invasion by directly targeting WNT-1

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