Dear Kevin, Thank you so much for this video! I am a third year medical student in the UK and am doing a lab-based dissertation for a semester. I am only 19 and have NO idea what I'm doing. My professor has just told me that we will be doing TSA on mice brain slices and being confused is an understatement. Moreover, I have absolutely zero clue how to write a 10,000 word dissertation on something I don't even know how to pronounce. This video really helped explain a lot of what im about to do tomorrow. Keep going! Regards, A very confused and sleepy student :)
Thanks for the support! Do note that I also have a live demo video that accompanies this, aiming to visualize the nuance & logistics of actually doing the technique. Good luck, and feel free to ping me back if you have follow-up Qs.
This is a good question without a simple answer. It can vary depending on the following factors: 1. antibody concentration in solution, 2. section thickness, 3. amount of antigen present in each section (more ubiquitous antigens suck out more antibody from solution), 4. level of solution, 5. speed/rpm/shaking force applied to the netted well(s), 6. how much the sections clump or attach to each other, 7. width/height of the sections relative to the wells and whether they fold easily or even too excessively, and 8. addition of reagents that aid antibody penetration into tissue (usually Triton x-100, tween-20, or other surfactants & tissue permeabilizers). There might even be other factors I'm not aware of. But, to give you at least some estimate: I've done well having 3-4 formaldehyde-fixed adult rat brain sections (50 um thick) per netted well, half full with solution, set to shake just enough to move the sections. This is using the netted wells depicted from Ted Pella, and the 6-well insert + basin fits ~8 mL to fill it to about 1/2 height before sections are added. The antibody concentration is usually 1:2000 (0.5 ug/mL) for antigens that aren't too ubiquitous; DeltaFosB primary antibodies works well enough at this amount whereas something found everywhere like a GABA receptor antibody would probably need to be between 1:100 and 1:500. I haven't tried varying the secondary and tyramide dilutions accordingly but really should at some point, but when I use either of those at 1:300-1:500 it usually suffices to label the target appropriately. One consideration is that I hemisect my brain tissue as each hemisphere could be considered a back up if the target is expressed equally in both hemispheres. As such, I see no need to stain intact (both hemisphere) sections when I can just use half-sections; the former would just eat up more antibody from solution and give extra info that I honestly wouldn't spend much time analyzing.
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Dear Kevin,
Thank you so much for this video! I am a third year medical student in the UK and am doing a lab-based dissertation for a semester. I am only 19 and have NO idea what I'm doing. My professor has just told me that we will be doing TSA on mice brain slices and being confused is an understatement. Moreover, I have absolutely zero clue how to write a 10,000 word dissertation on something I don't even know how to pronounce. This video really helped explain a lot of what im about to do tomorrow. Keep going!
Regards,
A very confused and sleepy student :)
Thanks for the support! Do note that I also have a live demo video that accompanies this, aiming to visualize the nuance & logistics of actually doing the technique. Good luck, and feel free to ping me back if you have follow-up Qs.
How many slices do you put into each netted well?
This is a good question without a simple answer. It can vary depending on the following factors: 1. antibody concentration in solution, 2. section thickness, 3. amount of antigen present in each section (more ubiquitous antigens suck out more antibody from solution), 4. level of solution, 5. speed/rpm/shaking force applied to the netted well(s), 6. how much the sections clump or attach to each other, 7. width/height of the sections relative to the wells and whether they fold easily or even too excessively, and 8. addition of reagents that aid antibody penetration into tissue (usually Triton x-100, tween-20, or other surfactants & tissue permeabilizers). There might even be other factors I'm not aware of.
But, to give you at least some estimate: I've done well having 3-4 formaldehyde-fixed adult rat brain sections (50 um thick) per netted well, half full with solution, set to shake just enough to move the sections. This is using the netted wells depicted from Ted Pella, and the 6-well insert + basin fits ~8 mL to fill it to about 1/2 height before sections are added. The antibody concentration is usually 1:2000 (0.5 ug/mL) for antigens that aren't too ubiquitous; DeltaFosB primary antibodies works well enough at this amount whereas something found everywhere like a GABA receptor antibody would probably need to be between 1:100 and 1:500. I haven't tried varying the secondary and tyramide dilutions accordingly but really should at some point, but when I use either of those at 1:300-1:500 it usually suffices to label the target appropriately.
One consideration is that I hemisect my brain tissue as each hemisphere could be considered a back up if the target is expressed equally in both hemispheres. As such, I see no need to stain intact (both hemisphere) sections when I can just use half-sections; the former would just eat up more antibody from solution and give extra info that I honestly wouldn't spend much time analyzing.