we recommend you to consider the optimal conditions each time.

Code No. 019-19741

Problem
No signal

Anti Iba1, Rabbit（for Immunocytochemistry）

Preparation : Purified by the antigen affinity chromatography fromrabbit antisera and prepared in TBS solution. Contains nopreservatives and stabilizers.
Specificity : R eactive with human, mouse and rat Iba1.
Working Concentration : Immunocytochemistry 1-2μg/mL	Immunohistochemistry（frozen section）0.5-1μg/mL
Storage : Keep at − 20℃.
After opening aliquot contents and freeze at − 20℃.
Package : 50μg（100μL）

Solution
Check the 1st antibody and2nd antibody, please add.

2nd antibody is not compatible Use a 2nd antibody against
rabbit IgG.

抗 Iba1，ウサギ（免疫細胞化学用）

Rabbit Anti Iba1 antibody is raised against synthetic peptide
corresponding to C-terminus of Iba1.
For Research Use Only. Not for use in diagnostic procedures ortherapeutic use.

Possible cause
1st and/or 2nd antibody
NOT added or enough.

Weak signal

In case of immunofluores- Select a suitable filter forcence method, a suitable
the using fluorescent dye.
fluorescent filter is notused.
Perfusion-fixation is not done. Perform perfusion-fixation.
The antigens are denatured. Do antigen retrieval-by using
A or B buffer.
（A）Citrate Buffer（pH 6.0）,
90℃, 9 min（B）TE buffer（pH 9.0）,
90℃, 9 min
The section is old or broken. Make new section. 50μm
thickness section works
well.

In case of Immunofluorescence method, the laser
intensity of fluorescencemicroscope is too weak.
Stain neurons The concentration of Antiand microglia Iba1 antibody is too high.

Adjust the laser intensity.

Recommended protocol（Immunohistochemistry, frozen section）
Rat or mouse was perfusion-fixed with 4% paraformaldehyde. replaced
sucrose, and prepared 50μm brain section by microtome.

The concentration of 2nd
antibody is too high.

Titrate the antibody to theoptimal concentration.
Recommended dilution
range is 1 : 500-1,000
Titrate the antibody to theoptimal concentration.

The reaction time of 2nd
antibody is too long.

Recommended reaction
time is 1-2 hour.

Wash : 0.3% TritonX-100 in PBS, 5 min×3↓
Blocking : 1% BSA, 0.3% TritonX-100/PBS, 2 hours, RT↓
Primary antibody : R abbit anti-Iba1（ 1：1000）, 1% BSA, and 0.3%
TritonX-100 in PBS, Over night, 4℃↓Wash : 0.3% TritonX-100 in PBS, 5 min×3↓, 1% BSA,
Secondary antibody : AlexaFluor®488 anti-rabbit IgG（1 : 1000）
and 0.3% TritonX-100 in PBS, 2 hours, RT↓
Wash : 0.3% TritonX-100 in PBS, 5 min×3↓
Mount

The antigens are denatured. Do antigen retrieval by using
A or B buffer.
（A）Citrate Buffer（pH 6.0）,
90℃, 9 min（B）TE buffer（pH 9.0）,
90℃, 9 min

Trouble Shooting
We confirm this product has as good quality as that of the previous lot
by immunostaining test. Since this product is polyclonal antibody, best
assay conditions vary depending on the activity of each lot. Therefore
—1/3—

High
background

Blocking is absent orinsufficient.

Extend the blocking
incubation time.
Try another blocking solution.（Wako recommends
PBS with 1% BSA, 0.3%
Tween-20 or 3% normalsserum of 2nd antibody’host）.

The concentration of 2ndantibody is too high.

Titrate the antibody to theoptimal concentration.

The reaction time of 2nd
antibody is too long.

Recommended reaction
time is 1-2 hour.

The section is old or broken. Make new section. 50μm
thickness section works
well.

—2/3—

ProblemHighbackground

Possible cause

Solution

The antigens are denatured. Do antigen retrieval by using
A or B buffer.
（A）Citrate Buffer（pH 6.0）,
90℃, 9 min（B）TE buffer（pH 9.0）,
90℃, 9 min
In case of immunoenzyme
method, reaction time ofsubstrate is too long.

Shorten reaction time.

In case of using biotinconjugated antibody,endogenous peroxidases
are active.

Inactivate endogenousperoxidases.
Before blocking, incubatewith 3% H2O2, 80%Methanol,-20℃, 20 min.

Staining clearer This antibody’s property
in rat than inmouse.

This product tends to stain
clearer in rat than in mouse（for unknown reasons）.

References :1）Imai, Y., Ibata, I., Ito, D., Ohsawa., K. and Kohsaka, S. : Biochem.
Biophys. Res. Commun ., 224, 855（1996）.
2）Ito, D., Imai, Y., Ohsawa, K., Nakajima, K., Fukuuchi, Y. and Kohsaka, S. :Brain Res. Mol. Brain Res ., 57, 1（1998）.
3）Ohsawa, K., Imai, Y., Kanazawa, H., Sasaki, Y. and Kohsaka, S. : J. Cell
Sci ., 113, 3073（2000）.
4）Sasaki, Y., Ohsawa, K., Kanazawa, H., Kohsaka, S. and Imai, Y. :Biochem. Biophys. Res. Commun ., 286, 292（2001）.
5）Kanazawa, H., Ohsawa, K., Sasaki, Y., Kohsaka, S. and Imai, Y. : J. Biol.
Chem ., 277, 20026（2002）.

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2003KA4