Institute of Science, Banaras Hindu University, Varanasi
Question
Asked 9th Apr, 2022
How can i preserve the mice brain so that i can utilise the same brain for immunostaining and western blotting?
I have to utilise treated mice brain for immunofluorescence and western blots. It is difficult and time consuming to generate treatment groups for these experiments. PFA fixation hampers western blots. Is there any other means of preservation so i can utilise same brain for western and immunostaining.
Popular answers (1)
Hello Krishna Singh Bisht,
As you know, to retain the cellular histo-architecture, you have to fixed the brain tissue as early as possible after its removal from the mouse. PFA cross-links the proteins in the tissue and changes their molecular weight in unpredictable and heterogenous ways. Although several researchers have tried antigen retrieval from PFA-fixed tissue, but the adverse impact of PFA fixation can easily be seen on the structure and antigenic nature of protein, which is one of the important aspect for the detection of protein using specific antibody in WB.
One more important point is that before sampling of brain tissue, you must be really careful what specifically you are trying to test. There is a lateralization of brain function in some structures of the brain, hence doing this could possibly lead to erroneous results. Just make sure that your specific question pertains to bilateral functionality of your specific area of interest.
In order to minimize the difficulties and time, it would be appropriate to collect the brain sample as given below:
Take 12 mice and divide into two groups; Group A (6 mice) and Group B (6 mice)
Group A (control group) and ---------------------------Group B (treatment group)
1. mouse : left side IHC, right side WB 1. mouse : left side IHC, right side WB
2. mouse : left side WB, right side IHC 2. mouse : left side WB, right side IHC
3. mouse : left side IHC, right side WB 3.mouse : left side IHC, right side WB
4. mouse : left side WB, right side IHC 4. mouse : left side WB, right side IHC
5. mouse : left side IHC, right side WB 5. mouse : left side IHC, right side WB
6. mouse : left side WB, right side IHC 6. mouse : left side WB, right side IHC
From the above experimental design, you would be in position to determine if using left vs. right makes a difference in your particular assay/hypothesis using statistical analyses. In addition, it will be easy for you to compare the protein changes in the cellular architecture of left side brain using IHC with that of corresponding brain tissue protein level using WB. Sometimes, it might make no difference at all but you don't want to inflate your chance of error by just randomly selecting given what you know or don't know about lateralization of brain structures.
In this way, you can collect the brain tissue for IHC as well as for WB from control and treated groups simultaneously in order to minimize the difficulties and time. In addition, in this way, you can overcome the issue of lateralization of brain structures.
Hope this helps
Best
3 Recommendations
All Answers (3)
Institute of Science, Banaras Hindu University, Varanasi
Hello Krishna Singh Bisht,
As you know, to retain the cellular histo-architecture, you have to fixed the brain tissue as early as possible after its removal from the mouse. PFA cross-links the proteins in the tissue and changes their molecular weight in unpredictable and heterogenous ways. Although several researchers have tried antigen retrieval from PFA-fixed tissue, but the adverse impact of PFA fixation can easily be seen on the structure and antigenic nature of protein, which is one of the important aspect for the detection of protein using specific antibody in WB.
One more important point is that before sampling of brain tissue, you must be really careful what specifically you are trying to test. There is a lateralization of brain function in some structures of the brain, hence doing this could possibly lead to erroneous results. Just make sure that your specific question pertains to bilateral functionality of your specific area of interest.
In order to minimize the difficulties and time, it would be appropriate to collect the brain sample as given below:
Take 12 mice and divide into two groups; Group A (6 mice) and Group B (6 mice)
Group A (control group) and ---------------------------Group B (treatment group)
1. mouse : left side IHC, right side WB 1. mouse : left side IHC, right side WB
2. mouse : left side WB, right side IHC 2. mouse : left side WB, right side IHC
3. mouse : left side IHC, right side WB 3.mouse : left side IHC, right side WB
4. mouse : left side WB, right side IHC 4. mouse : left side WB, right side IHC
5. mouse : left side IHC, right side WB 5. mouse : left side IHC, right side WB
6. mouse : left side WB, right side IHC 6. mouse : left side WB, right side IHC
From the above experimental design, you would be in position to determine if using left vs. right makes a difference in your particular assay/hypothesis using statistical analyses. In addition, it will be easy for you to compare the protein changes in the cellular architecture of left side brain using IHC with that of corresponding brain tissue protein level using WB. Sometimes, it might make no difference at all but you don't want to inflate your chance of error by just randomly selecting given what you know or don't know about lateralization of brain structures.
In this way, you can collect the brain tissue for IHC as well as for WB from control and treated groups simultaneously in order to minimize the difficulties and time. In addition, in this way, you can overcome the issue of lateralization of brain structures.
Hope this helps
Best
3 Recommendations
Johns Hopkins University School of Medicine
Hi Krishna,
What I can think of, take the brain out from the animal quickly, snap freeze in Liquid Nitrogen
or
Place the brain in a mold full of OCT and place the mold on Dry ice so that you can keep track of brain orientation till it freezes. After it is frozen on dry ice, you can transfer the mold in LN2 or leave at -86C till you cut the sections
Start cutting sections in Cryostat, Keep alternate sections and place directly in Lysis buffers for protein extraction, rest cryosections for IHC. Since they are frozen sections, you can fix after thawing the sections and no antigen retrieval needed for frozen sections
It will not going to affect your experiment if are quick to retrieve brain and place in LN2 or on Dry ice on mold. You can do both Western and IHC comfortably Only thing is that you need LN2 and Cryostat and OCT
Good luck
Bielefeld University
Hello,
in our Lab, we isolate the brain and divide it into hemispheres.
- Left hemisphere into PFA, 24 to 48 hours (depending on the brain size), then 30% Sucrose for cryoprotection, then freeze at -80°C. You can use this one for IHC
- Right hemisphere in Tissue Protein Extraction Reagent for protein isolation.
Please do not hesitate, if you have any further questions.
Best,
Tamara
1 Recommendation
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