3 replies to this topic

[madelingirly]

Dear Guys ,
In the beginning, I am very grateful for any one who will read my topic.

Secondly, I want to buy some culture wares for visualization of my cells under fluorescence or phase contrast microscope.
what I found due to reading some books and online:

*There are special type of culture slide such as
http://www.bdbioscie...y=4&mterms=true

*It is untreated.
I should treat the surface with Poly L lysine or collagenase as I am working with adherent cells
but I cant know the difference between these reagent and which on is the best.

* After culturing, Does the step of fixation is necessary, or adhesion to surface of my slide is enough.
* Shall I use the ordinary glass cover on my slide or use these precoated glass covers or precoat them by myself.
http://www.bdbioscie...rue&from=dTable

finally, does these steps differe if I will use a laser scanning confocal microscope?? has any one has experience with it and ready to share??

At the end I am deeply indebted for those who will share their thoughts, experience with me.

Best Regards

[doxorubicin]

Most adherent cells should grow on glass just fine with no poly L lysine treatment.  Some tricky cells (e.g. 293) might require poly L lysine so that they don't spontaneously detach.  Chamber slides are nice, since they will allow you to use less reagents for later processing steps.  In the old days, we used to just drop sterile cover slips into 6-well plates, and cells would attach just fine to the cover slips.  If doing confocal microscopy, the thickness of the coverslips may be crucial, so check with your local confocal expert prior to choosing cover slips.  If you are trying to visualize a fluorescent protein expressed by your cells, you won't need to fix the cells.  If you are going to be doing staining with antibodies, you will need to do a fixation/permeabilization step.  I personally like to use one that involves 3.7% formaldehyde (in PBS) at 37C for 10 minutes followed by 100% of -20C Methanol for 30 minutes.

[fysio lab]

doxorubicin, on 07 February 2012 - 09:55 PM, said:

Most adherent cells should grow on glass just fine with no poly L lysine treatment.  Some tricky cells (e.g. 293) might require poly L lysine so that they don't spontaneously detach.  Chamber slides are nice, since they will allow you to use less reagents for later processing steps.  In the old days, we used to just drop sterile cover slips into 6-well plates, and cells would attach just fine to the cover slips.  If doing confocal microscopy, the thickness of the coverslips may be crucial, so check with your local confocal expert prior to choosing cover slips.  If you are trying to visualize a fluorescent protein expressed by your cells, you won't need to fix the cells.  If you are going to be doing staining with antibodies, you will need to do a fixation/permeabilization step.  I personally like to use one that involves 3.7% formaldehyde (in PBS) at 37C for 10 minutes followed by 100% of -20C Methanol for 30 minutes.

Chamber slides are indeed very handy...but you need to know which type of CSLM you have first. Is it an upright or an inverted?

[scolix]

have a complete staining protocol.
try out both coating material, some cells have preferences.
check with local confocal microscope super user for additional in choosing coverslips vs slides