Hi!

I have some trouble with IHC staining c-Fos in the PVN and ARC area of the brain. The stainnig was not able to count the positive cells. And when I decrease the background color, the green cells turns to be dark too. So, I am not sure if I really stained the c-Fos.

I used the 20-week-old wild type mouse to do the IHC. Mouse were treated with 500ug/Kg Liraglutide or PBS (1.p.) 1.5hr before Anesthetize。

The frozen section was cut by 20um

1st antibody: Anti-c-Fos (Ab-2) (4-17) Rabbit pAb, code: PC05 dillute by 1:50

2nd antibody: Alexa fluor 488 goat anti-rabbit IgG[H+L] , Invitrogen A11034, dillute by 1:200

(Dillution by in 0.3%Triton X-100/1% BSA/1x PBS)

Here is my Protocol:

Step Frozen section:

1. Anesthetize: 1g/100ml Pentobarbital( 50mg/kg BW=0.005ml/g BW)

2. Transcardial perfusion with cold PBS for 3min at speed 7.5, and then 4% paraformaldehyde(PFA) for 3min

3. Fixation: 4% PFA overnight 24hr, 4℃ in dark. (40ml PFA 50ml Falcon tube)

4. Dissect the brain, cut the dissections including Interbrain and Hindbrain by ASI rodent brain matrix(Adult mouse, 30g,coronal),

5. Fixation: 4% PFA overnight 24hr, 4℃ in dark.

6. Equilibration: 10% (w/v)sucrose/PBS, 12-24hr, 4℃, in dark. (40ml Sucrose buffer)

7. 15% (w/v)sucrose/PBS, 12-24hr, 4℃, in dark

8. 20% (w/v)sucrose/PBS until the tissue is sunk, 12-24hr, 4℃, in dark

9. Place the tissue block onto a pre-labeled tissue base mold, Cover the entire tissue block with cryo-embedding media.

10. Slowly place the base mold containing the tissue block into liquid nitrogen until the entire tissue block is submerged into liquid nitrogen to ensure tissue is completely frozen(Can store the frozen tissue block at -80ºC until use)

11. equilibrate at -15~-20°C for approximately 15 minutes before attempting to section.

12. Section into 20μm(for slide), about 1.5mm from bregma

IHC steps

1. 0.3%Triton X-100/1x PBS: 5min 2X

2. Blocking: Circle sample by pen. Blocking solution 30min, RT, wet tissue

3. 1st antibody(1:50): 100ul/sample, 1 days, 4℃, cold room,dark condition

4. Wash: 0.1%Triton X-100/1x PBS, 5min, 3X

5. 2nd antibody(1:200): 100ul/sample, 3h, RT, under dark condition.

6. Wash: PBS 5min, 3X

7. Counterstain with DAPI solution, 30min

8. Wash: PBS, 5min, 3X

9. Aspirate the PBS around the tissue, dry a few minutes

10. Antifade: 20μl in the middle of tissues

11. Cover glass, nail gel, dark condition, overnight

Could you please tell me where is wrong in my protocol?

Thank a lot.