Products

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

BCIP-NBT Solution Company A
Red AP chromogenic substrate
BCIP-NBT_15.PNG
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
BCIP-NBT_4.png BCIP-NBT_5.png
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

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

Company D
BCIP-NBT (Substrate Set)
BCIP-NBT Solution
BCIP-NBT_6.png
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.

Comparison: Detection sensitivity by dot blotting

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

BCIP-NBT_7.png

Application 3: In situ hybridization (ISH)

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
BCIP-NBT_8.png BCIP-NBT_9.png
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)

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.


Reaction time

5 minutes
BCIP-NBT_10.png
10 minutes
BCIP-NBT_11.png
15 minutes
BCIP-NBT_12.png
30 minutes BCIP-NBT_16.png
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)

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.

BCIP-NBT_20.png

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.

Ordering information

Product Name Grade Storage Catalog Number PKG Size Price
BCIP-NBT Solution (Ready To Use) SP Refrigerate 19880-84 100 mL e-Nacalai

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Related product

Product Name Grade Storage Catalog Number PKG Size Price
Blocking One Histo SP Refrigerate 06349-64 50 mL e-Nacalai
HistoVT One (10x, pH 7.0) SP Room temperature 06380-76 100 mL
06380-05 500 mL
4%-Paraformaldehyde Phosphate
Buffer Solution
SP Refrigerate 09154-14 5 x 10 mL
09154-56 100 mL
09154-85 500 mL