iPS cell

BCIP-NBT Solution	Company A Red AP chromogenic substrate

Experiment conditions
Cell :	iPS cell (01B7 line*), feeder cell (SNL)
Fixation :	4%-Paraformaldehyde Phosphate Buffer Solution (#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

Added BMP-2 (100 ng/mL) Cultured for 3 weeks	No BMP-2 Cultured for 3 weeks

Experiment conditions
Cell :	MC3T3-E1 Cell
Fixation :	4%-Paraformaldehyde Phosphate Buffer Solution (#09154) 10 minutes in room temperature
Staining :	BCIP-NBT Solution (Ready To Use), 30 minutes at room temperature
Detection :	Optical microscope (Olympus # BX50) by differential interference, 50x magnification

Company D BCIP-NBT (Substrate Set)	BCIP-NBT Solution

Experiment conditions
Sample :	Protein from mouse brown adipose tissue (From left 30 μg, 10 μg, 3 μg, 1 μg)
Blotting :	5% skim milk/tTBS at room temperature for 1 hour.
Primary antibody :	Anti-UCP1 antibody (Rabbit) (Self-preparation) 2,000x diluted Diluted using 5% skim milk/tTBS, refrigerated overnight.
Secondary antibody :	Anti-Rabbit IgG (Fc), AP Conjugate(Promega #S373B) 5,000x diluted Diluted using 5% skim milk/tTBS, left at room temperature 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 order hand, 1 bottle type of BCIP-NBT Solution (Ready To Use) is convenience and results as good as others.

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

Compare with other brands, BCIP-NBT Solution has equal or better sensitivity.

Detecting IX type collagen (α1) 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).

Antisense strand	Sense strand

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 (#06349-64) at room temperature for 10 minutes
Primary antibody :	Anti-Digoxigenin-AP, Fab fragments from sheep (Roche #11093274910) 1:2,000 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 (Diagnostic BioSystems #K002) and Entellan® new (Merck #107961), observed by Zoom Stereomicroscope MF2.6x (AXEL #1-1925-02), microscope MF 50x (Olympus #BX50)

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

Reaction time

5 minutes	10 minutes
15 minutes	30 minutes

Experiment conditions
Tissue :	Small intestine paraffin section from mouse
Antigen activation :	HistoVT One (10x, pH 7.0) (#06380) at 90°C for 20 minutes
Blocking :	Blocking One Histo (#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) 1:1,000Used 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 (Diagnostic BioSystems #K002), observed by microscope MF 25x (Olympus #BX50)

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.

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.