Antibody Guarantee - Immunohistochemistry
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No "brown" staining
Possible causes:
• Were the primary and secondary antibodies added?
• Concentration of primary antibody may be too low. Increase the concentration by a factor of 2 - 3.
• Make sure the H2O2 was added to the DAB solution. Use a fresh stock bottle of H2O2 .
• Was the enyzme pretreatment step performed?
• Slides left too short a time in the DAB solution.
• Is the tissue type known to express the antigen?
• Pretreatment was too long and the epitope was destroyed. Reduce the incubation time.
• Were the antibodies stored at the correct temperature? Antibodies sold in solution will be inactivated by freezing, while antibodies sold lyophilized are best stored frozen after they are reconstituted.
• Is the tissue of the correct species origin?
"Brown" staining too light
Possible causes:
• Primary antibody concentration too low. Increase the concentration by a factor of 2 - 3.
• Increase the incubation time with the primary antibody.
• Slides left too short a time in the DAB solution.
• Level of expression may be low for the specific tissue sample.
• How old are the reagents/kit? Check expiration date on vial(s).
• Enhance staining by placing slides in 0.5% cupric sulfate in 0.15 M NaCl for 5 minutes. Rinse in de-ionized H2O, then counterstain with hematoxylin.
Sections are too brown or entire section is brown
Possible causes:
• Primary antibody concentration too high. Decrease the concentration by a factor of 2 - 3.
• Slides left in the DAB solution too long. Shorten the incubation time in DAB.
• Tissue was not properly blocked. Increase the incubation time with the blocking serum and/or increase the concentration of blocking serum by a factor of 3. If BSA is used, be sure it is fatty acid-free.
• Tissue was not properly blocked. Increase the incubation time with the blocking serum and/or increase the concentration of blocking serum by a factor of 3. If BSA is used, be sure it is fatty acid-free.
• Washing steps in between incubations with antibody were shortened or omitted altogether.
• Preincubation with H2O2 blocking solution was not performed.
• H2O2 is old. New reagent should be obtained.
• Cross-reactivity between the secondary antibody and proteins in the tissue may be occurring. Add one or both:
- 1% normal rabbit serum (Calbiochem Cat. No. NS01L) to the blocking solution.
- 0.01% TRITON® X-100 (Calbiochem Cat. No. 648462) to the primary antibody solution (caution: this may also reduce specific binding of some antibodies).
Debris found on the slides
Possible causes:
• DAB is old and beginning to precipitate out of solution. Filter DAB solution before using.
• Tissue sections are of poor quality. New sections should be obtained.
• Xylene is dirty and should be changed.
No counterstaining or counterstaining is too light
Possible causes:
• Increase time left in the Hematoxylin solution.
• Use a "bluing" reagent to enhance the staining.
Counterstain is too dark (no contrast is seen between the counterstain and DAB)
Possible causes:
• Sections left in hematoxylin solution too long. Reduce the incubation period.
• Insufficient incubation with DAB. Increase the incubation time in DAB.
• Try an alternate histological counterstain such as methyl green.
• Place sections in 70% ethanol with 1% HCl (1N) until stain lightens.
Staining pattern not what is expected (cytoplasmic as well as nuclear)
Possible causes:
• Tissue was over digested with the enzyme pretreatment step. Reduce the duration of the incubation or the concentration of enzyme used.
• Sections were improperly blocked with the serum blocker. Increase incubation time for the blocking step or increase the concentration of blocker used.
• Excess primary antibody was used. Decrease the concentration of primary antibody. Cross-reactivity between the secondary antibody and proteins in the tissue may be occurring. Add 1% normal rabbit serum to the blocking solution or reduce the amount of secondary antibody by a factor of 3.
If the problem you are observing is not accurately described or remedied by any of the above suggestions please let us know using this form
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