Nacalai USA, Inc. | Product | BCIP-NBT Solution (Ready To Use)

BCIP-NBT Solution (Ready To Use)

A ready to use solution of chromogenic substrate of alkaline phosphatase (AP). It can be used in a wide range of applications, such as detection of alkaline phosphatase (AP) bound by antigen-antibody reaction and endogenous alkaline phosphatase (AP) of stem cells and osteoblasts, in blotting and tissue or cell staining.

Ready to use
Can be used in wide range of applications such as immunochemical staining or endogenous AP staining.
Can be used in in situ hybridization (ISH), as it is tested for DNase and RNase.

Application 1: Endogenous AP staining of iPS cells and osteoblasts

iPS Cell

Experiment Conditions:

Cell	iPS cell (01B7 line*), feeder cell (SNL)
Fixation	4%-Paraformaldehyde Phosphate Buffer Solution (Cat. No.: 09154)10 minutes at room temperature
Staining	60 minutes at room temperature
Detection	(Upper) Scanner (Seiko Epson)(Lower) Optical microscope (Olympus #BX50), 10X magnification

* Takahashi, K. et al. Cell. 2007, 131(5), p. 861-872

Osteoblasts

Experiment Conditions:

Cell	MC3T3-E1 Cell
Fixation	4%-Paraformaldehyde Phosphate Buffer Solution (Cat. No.: 09154), 10 minutes at room temperature
Staining	BCIP-NBT Solution (Ready To Use), 30 minutes at room temperature
Detection	Optical microscope (Olympus #BX50) by differential interference, 50X magnification

Application 2: Western blotting detection of UCP1 protein from mouse brown adipose tissue

Experiment Conditions:

Sample	Protein from mouse brown adipose tissue (From left 30 ug, 10 ug, 3 ug, 1 ug)
Blotting	5% skim milk/tTBS at room temperature for 1 hour.
Primary Antibody	Anti-UCP1 antibody (Rabbit) (Self-preparation) 2000x diluted. Diluted using 5% skim milk/tTBS, refrigerated overnight.
Secondary Antibody	Anti-Rabbit IgG (Fc), AP Conjugate (Promega #S373B) 5000x diluted. Diluted using 5% skim milk/tTBS, left at room temperarture for 1 hour.
Detection	1 minute for each staining solution

User evaluation:

BCIP-NBT (Substrate Set) from Company D is 2 bottles type which takes time to making our own buffer. On the other hand, 1 bottle type of BCIP-NBT Solution (Ready To Use) is convenience and results as good as others.

Data courtesy of Professor Goto, Tsuyoshi, Department of Food and Biological Sciences, Graduate School of Agriculture, Kyoto University.

Comparison: Detection sensitivity by dot blotting

Compare with other brands, BCIP-NBT Solution (Ready To Use) has equal or better sensitivity.

Application 3: In situ hybridization (ISH)

Detecting IX type collagen (alpha1) mRNA from fetal mouse paraffin embedded section. The product can be used for high sensitivity detection, such as is required with in situ hybridization (ISH).

Experiment Conditions:

Tissue	Paraffin section from fetal mouse (16.5 days) for ISH (Genostaff #SMPS-08-5)
Hybridization	RNA probe for DIG tag mouse Col9a1 ISH (Genostaff #MP-C-023)
Blocking	Blocking One Histo (Cat. No.: 06349-64) at room temperature for 10 minutes
Primary Antibody	Anti-Digoxigenin-AP, Fab fragments from sheep (Roche #11093274910) 1: 2000, Diluent: tTBS/Blocking One Histo in 19:1 ratio
Staining	BCIP-NBT Solution (Ready To Use), 4 hours at room temperature; Counterstain: Kernechtrot dye, 10 seconds at room temperature
Observation	Enclosed by CC/Mount (Doagnostic BioSystems #K002) and Entellan new (Merck #107961), observed by Zoom Stereomicroscope MF2.6x (AXEL #1-1925-02), microscope MF 50x (Olympus #BX50)

Application 4: Immunohistochemical staining

Detecting PCNA from mouse small intestine paraffin embedded section. if there is insufficient sensitivity, extending reaction time can result in good sensitivity.

Experiment Conditions:

Tissue	Small intestine paraffin section from mouse
Antigen Activation	HistoVT One (10x, pH 7.0) (Cat. No.: 06380) at 90C for 20 minutes.
Blocking	Blocking One Histo (Cat. No.: 06349-64) at room temperature for 10 minutes
Primary Antibody	PCNA Antibody (Novus #NB100-456) 1: 500, Diluent: tTBS/Blocking One Histo in 19:1 ratio
Secondary Antibody	Goat anti-Rabbit IgG (H+L) Secondary Antibody [Alkaline Phosphatase] (Pre-adsorbed) (Novus #NB7181) 1: 1000. Used tTBS as an antibody diluent
Staining	BCIP-NBT Solution (Ready To Use), reacted at room temperature for time shown above
Observation	Enclosed by CC/Mount (Doagnostic BioSystems #K002), observed by microscope MF 50x (Olympus #BX50)

Measurement principle

BCIP, a chromogenic substrate for alkaline phosphatase (AP), produces an indigo color by reaction. In the same reaction, NBT is reduced and turns into an insoluble purple precipitate.

Usage

Procedure for staining endogenous alkaline phosphatase (AP). For other applications of usage, please refer product instruction.
1. Fix cells with a fixing solution such as paraformaldehyde
2. Wash fixing solution with water.
3. Pour BCIP-NBT Solution slowly, and leave at room temperature to react. (For 35mm dish, please use 1mL of BCIP-NBT Solution)
4. When an appropriate stained image is obtained, wash with water to stop the reaction.
5. Observe it enclosed or after air drying.