Immunofluorescence Staining

Immunofluorescence Staining of

Frozen Sections

1. Samples and Materials

4%Paraformaldehyde; PBS/NS; fresh mammalian tissues/cells; OCT; sucrose; H2O2;

TritonX-100; blocking solution; the first, second (or third) antibodies; mounting medium;

cryotome; travenol infuser; tinfoil; -80 freezer; coverslips/microscopic slides;

37℃incubator; microtome blades;

2. Protocol

1). Perfusion: perfuse the mice models with cold PBS/NS via the systemic blood (or

pulmonary) circulation, depending on the purpose of your experiment, till the effluent liquid appears clear

2). Isolate target tissues or organs and put them into 4%Paraformaldehyde at 4℃

overnight; (Well fixation is very important!)followed by10, 20, 30% sucrose density-gradient dehydration (or simply 30% only) and ready for the next step when samples sink to the bottom

3). Dry the residual sucrose liquid on samples with paper and put them into tin foil

boxes; embed samples in the OCT compound; mark their side(s) and then put into -80 freezer

4). Cut the tissues/organs (turn on the machine in advance)

5). Incubate the slides for half to one hour at 37℃; followed by rinses with PBS/NS, 5

min * 3 (Very important: the samples can NOT be dried after this step!!!)

6). (optional step) Permeabilization; rinse or incubate the sections with PBS/NS, 5 min * 3

7). (optional step) 3%H2O2 for 10min or 0.3% H2O2 for 30min, at RT, depending on tissues or organs (e.g. liver and kidney are rich in endogenous peroxidase and

avidin/biotin…); r inse or incubate the sections with PBS/NS, 5 min * 3

8). Blocking (it would be the best to use the second antibody and blocking solution from the same species); 30min, RT; remove the blocking buffer but NO any buffer

wash/rinse! (Very important!!!)

9). Incubate the samples in the first/primary antibodies, at 4℃, overnight

10). Decant the first/primary antibody solutions and rinse/wash the slides, 5 min * 3

11). Incubate the samples in the secondary antibodies for 2hs, at RT

12). Decant the secondary antibody solutions and rinse/wash the slides, 5 min * 3

13). (optional step) DAPI to dye nuclei for 5min at RT

14). Decant the DAPI solution and rinse/wash the slides, 5 min * 3

15). Mounting: glycerol or other mounting media (if the mounting medium includes DAPI, ignore the step 14 and 15)

16). (optional step) Seal coverslips with nail polish to prevent drying and movement of the samples/sections

17). Ready for imaging (the slides could be stored in dark at 4℃ temporarily)

Note: 1. Please always make sure all the solutions cover the entire samples/sections!!!

2. Please keep the slides in dark when necessary!

3. Be sure to use the first/primary antibodies, the secondary antibodies (or the third…) and blocking solutions APPROPRIATELY!!! (Multicolor staining can be performed either simultaneously, or sequentially)

4. This protocol is flexible and only for a reference. Optimal methods should be determined by the users.

11-12-2014