Confocal Resources

Fluorescent Probe Selection

Please review our equipment page to determine which fluorescent probes are compatible with the laser lines on our systems.

You may use the following spectral tools below, to help you with your selection. If you need further assistance, please contact Susan, staurovsky@uchc.edu, 860-679-4686.

 

Sample Preparation

Please note, the 780 and 880 Zeiss confocals are on inverted microscopes. Because of this, CCAM requires your samples to be sealed when mounted between a slide and cover glass. This will not only protect your sample but it will also protect our objectives and microscopes.

 

Sample Thickness
Widefield microscope sections typically range from 4-10 microns. Confocal sections often range from 10-40 microns.

 

Coverslip
Please use a #1.5, or 170 micron thick, glass coverslip.  All the Zeiss objectives are corrected to be used with this material and thickness.

 

Sealing Samples
Most commonly nail polish is used to seal slides, but it is toxic to live cells. Please refer to Coverslip Sealing for a variety of options.

CoverGrip  designed with ingredients that do not leach into aqueous mounting medium and affect specimen fluorescence.

 

Mounting Media
Mounting media should have the same refractive index or be very close to Zeiss’ immersion oil which has a RI of 1.518.

It is highly recommended to use a mounting media that contains an anti-fade agent such as n-propyl gallate.

The type of mounting media you use will depend on which probes you are trying to visualize. Glycerol based mounts, ProLong Diamond, are better for preserving structural information while mounts that harden can disrupt cellular structure. You can seal these types of  media before they harden and they will work equally as well and maintain the structures.

Mounting media can affect probes differently, so please make educated choices based on which probes you will be using. For example, Vectashield is not compatible with Alexa 647 and other far red dyes but works well with blue/green dyes. Likewise dated bottles of glycerol can result in unexpected staining results such as the nucleus appearing as background, as pictured below.

figure 4

Jonkman, J., Brown, C.M., Wright, G.D. et al. Tutorial: guidance for quantitative confocal microscopy. Nat Protoc 15, 1585–1611 (2020). https://doi.org/10.1038/s41596-020-0313-9

 

Homemade Mounting Media

MOWIOL Mounting Media

Jackson ImmunoResearch Anti-fade Mounting Medium

 

DAPI
Do not use mounting media that contains DAPI since premixed mounting solutions with DAPI tend to increase background fluorescence. It is best to first stain with DAPI and then mount your sample separately.

 

Live Cell Imaging Supplies

Cellvis – Glass Bottom Dishes

Fisher Scientific – Thermo Scientific Nunc Glass Bottom Dishes and Thermo Scientific Nunc Lab-Tek and Lab Tek II Chamber Slides

Ibidi – Chambered Coverslips

Mattek – Glass Bottom Dishes

Nunc Lab-Tek II Chambered Coverglass

Wilco – Glass Bottom Dishes