Name: In situ hybridization for mRNA kit
SKU: BCISHM1
Price: 1065.00 USD
Availability: InStock

In situ hybridization for mRNA kit

$1,065.00

Item Cat No.: BCISHM1

Application: Cryosection and FFPE section

Reactivity: Tissue

In situ hybridization for mRNA kit allows in situ detection and quantitation of mRNA expression at single cell level on cryosection or FFPE section of human and rodent tissues.

1 Kit (50 slides)

In situ hybridization enables the detection and precise localization of a specific nucleic acid sequence within an individual cell. The nucleic acid sequence is bound specifically in a tissue section by complementary base pairing, that is, hybridization, with a detectable nucleic acid segment called a probe.

BiCell Scientific Inc has developed and validated an in situ hybridization kit for mRNA detection based upon DIG-labeled oligonucleotide probe. The kit can be used for both cryosections and FFPE sections of rodent or human tissues. BiCell Scientific Inc has developed an algorithm to calculate the GC:AT ratios of oligonucleotides in order to maximize the hybridization efficacy. Each kit includes a validated oligonucleotide probe that has been selected for the target gene.

The kit contains:

1 L of DEPC treated water
10 ml of Permeabilization buffer (Proteinase K, NaCl, Phosphate buffered saline)
10 ml of Hybridization buffer (SSC, DTT, t-RNA, dextran sulfate) (proprietary)
500 μl of DIG-oligonucleotide probes (2 probes, each made of single-strand DNA) (10 μM) (sense strand probe for negative control; antisense strand probe for detection)
500 ml of Wash buffer (SSC, formamide, Tween-20)
100 ml of DIG-Block buffer (bovine serum, Tween-20, NaCl and Tris buffered saline)
500 ml of DIG-Wash buffer (Tween-20, NaCl, and phosphate Tris saline)
50 μl of anti-DIG primary antibody
50 μl of alkaline phosphatase conjugated secondary antibody
10 ml of Color developing buffer (NBT/BCIP, levamisole, Mg⁺⁺, NaCl and Tris buffered saline)
50 ml of Color stopping buffer (EDTA and Tris buffered saline)
1 ml of Mounting buffer (Mowiol and Tris buffered saline)

Store at -20^oC

For Research Use Only. Not for use in clinical diagnostics.

Thaw kit content to room temperature.
Deparaffinize FFPE sections with Xylene.
Rehydrate sections with decreasing concentrations of ethyl alcohol (100% to 70%) in DEPC-water.
Wash slides 2 times with DEPC-water.
Drain slides and overlay each section with 200 μl of Permeabilization buffer. Place slides in a wet chamber. Incubate the slides at 37^oC for 1hr.
Re-fix the slides in 4% PFA in PBS at room temperature for 30min.
Dehydrate the slides by passing through increasing concentrations of ethyl alcohol (70% to 100%) in DEPC-water. Air-dry the slides.
Pre-hybridize the slides with 200 μl of Hybridization buffer. Place slides in a wet chamber. Incubate the slides at 42^oC for 1hr.
Drain Hybridization buffer from the slides and overlay each section with 200 μl of Hybridization buffer containing 5 μl (50 pmol) of DIG-oligonucleotide probe. Heat the probe to 65^oC for 5min to linearize probe and chill on ice before adding to Hybridization buffer.
Incubate sections at 50°C overnight in a humid chamber.
Drain Hybridization buffer from the slides. Overlay each section with 200 μl of Wash buffer.
Incubate sections at 50°C for 30min in a humid chamber. Repeat washing step three more times.
Wash slides one time with 1xPBS at room temperature.
Drain slides and overlay each section with 200 μl of DIG-Block buffer containing 2 μl of anti-DIG primary antibody.
Incubate the slides in the wet chamber at room temperature for 2hr.
Take slides out of the wet chamber, drain off antibody, and wash three times with the DIG-Wash buffer.
Drain slides and overlay each section with 200 μl of DIG-Block buffer containing 2 μl of alkaline phosphatase conjugated secondary antibody.
Incubate the slides in the wet chamber at room temperature for 1hr.
Take slides out of the wet chamber, drain off antibody, and wash three times with the DIG-Wash buffer.
Drain Wash solution against a towel. Cover slide with 200 μl of Color developing buffer and incubate for 30min-5hrs until optimal color is achieved.
Stop the color development by draining slides and adding 200 μl of Color stopping buffer.
Drain Wash solution against a towel. Add 20 ul of Mowiol solution to the section on each slide. Place a cover glass onto the section and apply a small pressure to remove any trapped air bubble.
Let Mowiol solution dry at room temperature for 1hr. Store glass slides at 4^oC for less than 1 month.

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