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Problem 1: lack of staining Possible Cause Countermeasure Lack of antigen Check protein expression by in situ hybridization.
Due to improper storage, antibodies do not work as stored in the instruction manual. In general, the antibody is aliquoted in small portions, sufficient for a single experiment. Store these antibodies in a manually defrosted refrigerator (-20 to -70 °C) to avoid repeated freezing and thawing.
Primary or secondary antibody inactivation independent test reporting system to assess the usefulness of the reagent.
Insufficient fixation of the tissue Try to increase the fixed time or change the fixative.
Over-fixation of tissue reduces the duration of steps after infiltration or fixation. If infiltration is unavoidable, the antigen is not masked by the antigen retrieval agent.
Primary and secondary antibodies are not compatible with secondary antibodies that interact with primary antibodies. For example, if the primary antibody is from a rabbit, use an anti-rabbit secondary antibody.
The epitope has been altered during fixation or embedding attempts to restore immunoreactivity by various antigen retrieval techniques.
Tissues are embedded at 58°C or below.
Antigen repair does not work to increase the processing time or change the treatment solution.
The reagents are missing or in an incorrect order. Repeat the staining process. Confirm that the correct reagents are used and add them in the correct order.
Question 2: High Background Probable Causes Countermeasures High-titration of antibodies at the concentration of primary or secondary antibodies to determine the concentration of zuijia required to promote the specific responses of primary and secondary antibodies. .
Hydrophobic interactions of antibodies and proteins in tissues reduce the ionic strength (especially monoclonal antibodies) of antibody dilutions.
Non-specific binding of primary or secondary antibodies to tissue uses a blocking step (typically using 1% BSA and 10% normal donkey serum) prior to incubation with the primary antibody. Skim milk powder is also an option.
Nonspecific binding of the secondary antibody uses antibodies with cross-reactive IgG removed.
The tissue dries during the staining process to avoid drying out the tissue.
The background caused by the ionic interaction increases the ionic strength of the diluent.
Question 3: Cell/tissue morphology is destroyed Possible causes Countermeasures Antigen repair methods are too rigorous Experimental conditions are determined based on experience. Immunoreactive tissue sections that can retain tissue morphology and restore antigen are detached from the slide for a fixed period of time. Another fixative is determined empirically.
The tissue section is torn or folded. Re-slice the bubbles under the slice, or ignore the damaged area when analyzing the results.
The difference in resolution of the tissue morphology cuts out thinner tissue sections. Ice crystals may destroy the shape of frozen sections.
Insufficient fixation can cause tissue or cells to be damaged for a fixed period of time during staining. Increase the fixative/tissue ratio. Cut out smaller tissues for more thorough infiltration.
Tissue autolysis leads to staining of necrotic debris for a prolonged period of time and proportion. Consider using crosslinked fixatives.
Problem 4: Improper dyeing Possible cause Countermeasures Improper fixing method Try another fixing agent, or extend the fixing time.
Antigen remediation may improperly try another antigen retrieval condition.
The electrostatic charge of the antigen is changed to try to adjust the pH or cation concentration of the antibody dilution.
Fixed delays cause antigen diffusion to quickly fix tissue. Try cross-linking fixatives instead of organic fixatives.
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Analysis of common problems in IHC/ICC experiments
Technically speaking, IHC and ICC experiments are not difficult, however, there are so many variables in each experiment that need to be identified and optimized. If the bad luck, the experimental results are not very good, then we may have to do troubleshooting to see where the problem lies. The following table lists the common problems of the IHC/ICC experiment analyzed by Shanghai Yuan Mu senior technology, and the corresponding countermeasures.