Anti-LIM-1 Antibody

C-terminal portion of frog LIM-1 protein.

Anti-LIM-1 Antibody detects level of LIM-1 & has been published & validated for use in IF, IP, WB, IC, IH(P).

Immunohistochemistry: 1:200-1:1,000 (Fish and whole mounts 1:500) Recommended fixative MEMFA.

Our photo was produced from immersion fixation of chicken embryos in cold 4% PFA for 15-30 minutes, then sectioning was performed on a cryostat.

Works on paraffin embedded sections when sections are either lightly PFA fixed or fixed with acetone, ethanol or fixative suggested below.

SUGGESTED IMMUNOHISTOCHEMISTRY PROTOCOL FOR AB3200

The best fixative is MEMFA. 10X stock solution for MEMFA: 1 M MOPS, 20 MM EGTA. 10 mM MgSO4, 38% Formaldehyde. Fixation 1 hour, 2 x 15 min methanol. Following this protocol embryos may be stored in methanol at -20°C indefinitely or immediately embedded in paraplast. Best results on paraffin sections 6-10 micron thick.2) Staining following deparaffinization in xylene and a row of alcohol wash two times in water. Block in 2% Boehringer Mannheim reagent in 0.1 M maleic acid, pH 7.5, 150 mM NaCl for one hour at room temperature.3) Dilute the AB3200 in same blocking reagent and incubate overnight at 4°C or for at least 5 hours. Wash three times in PBS, 10 min each.4) Incubate with alkaline phosphatase conjugated secondary antibody (for example Chemicon Catalog Number AP132A. Develop with BCIP/TNBT (Chemicon Catalog Number ES007-100ML).5) For sections always use Digene silanated slides or Superfrost plus from Fisher as some times you may need to boil sections in 6 M urea for 5-6 min. in microwave at 80% power following deparaffinization to increase signal. That is especially useful if tissues were fixed in PFA.6) Sometimes it is necessary to predeplete antibody on hyperfixed embryos to lower background (especially for staining species other than frog and for whole-mounts). Procedure: hyperfix frog or fish embryos in MEMFA for 36-48 hours at RT 30-50 embryos. Wash 2 X in methanol (see above). Apply antibody in final dilution in blocking reagent for one hour on rocking table. Collect super and apply to your embryos or sections. For whole mounts use the following procedure: after fixation, methanol and PBS; block in PBST + serum (PBS + 2 mg/mL BSA + 0.1% Triton X100 + 10% animal serum) one hour room temperature. Add first antibody in PBST + serum and incubate over night at 4°C. Wash in PBST (no serum) 4 times for 2 hours. Add secondary diluted in PBST + serum over night at 4°C. Wash in PBST four times for 2 hours. Develop staining.

Do not use tissues fixed overnight in PFA the antibody will not work.

Immunocytochemistry: 1:500 on P19 cell line, lightly fixed (2% PFA) 5-15 minutes, permeabilized with 0.1% triton X-100 or methanol ( 5′ air dry).

Western blot: 1:3,000-1:6,000

Immunoprecipitation: 1:200

Immunofluorescence 1:100

Optimal working dilutions must be determined by the end user.

Research Category
Neuroscience

Research Sub Category
Developmental Neuroscience

Neuronal & Glial Markers

Recognizes LIM-1. This antibody does not appear to cross react with LIM-5 (the closest homologue of LIM-1 in frog) on tissue sections but does cross react with LIM-5 in Western blot and immunoprecipitation.

Format: Purified

Purified immunoglobulin

Maintain at 2-8°C in undiluted aliquots for up to 6 months.

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