What fixative is used for immunofluorescence?

What fixative is used for immunofluorescence?

Aldehyde-based fixatives such as formaldehyde, formalin (a mixture of dissolved formaldehyde with a lower percentage of methanol), and glutaraldehyde are most commonly used. For most antibodies, CST recommends fixation with 4% formaldehyde (IF Standard protocol).

Can you do IHC on frozen sections?

Immunohistochemistry Protocol. When staining cryostat sections stored in a freezer, thaw the slides at room temperature for 10-20 minutes. For fluorescent IHC staining of frozen tissue sections using R&D Systems antibodies, it is recommended to incubate overnight at 2-8 °C.

How do you fix frozen tissue sections?

Fix the tissue sections with a suitable fixative. One of the commonly used fixation methods for frozen tissue sections is to immerse the slides in pre-cooled acetone (-20°C) for 10 min. Pour off the fixative and allow acetone to evaporate from the tissue sections for < 20 min at room temperature.

How do you fix a frozen slide?

Fix slides by immersion in cold acetone (-20°C) for 2 minutes or other suitable fixative (e.g. alcohol, formal alcohol, formalin, etc.), air dry at RT and proceed to staining. Alternatively, the frozen section slides can be stored for a short period of time at -70°C in a sealed slide box.

How do you fix cells for fluorescence microscopy?

There are a number of fixation methods suitable for fluorescence microscopy that fall into two basic categories: aldehyde fixatives and alcohol fixatives. Organic solvents such as alcohols and acetone remove lipids and dehydrate the cells, while precipitating the proteins on the cellular architecture.

What is OCT in frozen section?

Tissue-Tek® O.C.T. Compound is used to quickly embed fresh tissue specimens for frozen sections using a cryostat. O.C.T stands for optimal cutting temperature. The high viscosity of Tissue-Tek O.C.T Compound results in fast freezing for optimal section quality.

Is antigen retrieval necessary for frozen sections?

The absence of a formaldehyde based fixative eliminates the need for an antigen retrieval step. However, if frozen tissue or cytological specimens have been fixed in formalin, antigen retrieval can be attempted, although the friable nature of the specimens, in particular brain tissue, may compromise the success.

How do you remove frozen tissue from Oct?

Hi Benoit, You can just let the OCT thaw at room temperature then blot away the melted OCT. To speed up the thawing process carefully cut away as much of the OCT as you can with a scalpel or razor blade and then let it thaw.

How do you mount a frozen section?

After the tissue is frozen, place it in dry ice and move to -80° C until ready for cutting. Embed tissue in OCT compound by slowly layering the compound so that the tissue does not thaw. Move the embedded tissue directly into the cryostat and use OCT medium to mount it to the chuck.

Why is blocking buffer used in immunofluorescence?

Blocking. Blocking is an important step for minimizing unspecific binding of the primary antibody within the cell. To achieve this, proteins from Bovine Serum Albumin (BSA), milk powder or serum can be used.

Can you do immunofluorescence on paraffin embedded sections?

The technique can be applied for other tissue types, both paraffin-embedded sections and cryosections. Although mice have been used in our experiments, this protocol can also be applied on tissues from other species. Figure 5. Immunofluorescence staining for different mouse tissues using our protocol.