JLA Immunofluorescence of BrdU

5-Bromodesoxiuridine (BrdU): BrdU (#B5002, SigmaAldrich) was diluted in

sterile NaCl 0,9% and administered at a dose of 50mg/kg intraperitoneally (i.p.). To
study proliferation during 24h from LPS administration, BrdU was incorporated after
5h, 12h and 22h from LPS administration. To study proliferation from 24h to 72h, BrdU
was incorporated at 24h, 36h, 48h, 60h and 70h after LPS administration.

Immunofluorescence: Mice from P60 to P160 were deeply anesthetized with

ketamine and xylazine cocktail and transcardially perfused with PBS1X followed by 4%
paraformaldehyde (PFA) in PBS1X (Phosphate buffered saline #p4417 Sigma). Brains
were post-fixed overnight at 4ºC in 4% PFA and embedded in saccharose 30%. 50μm
coronal sections were cut by cryotome and preserved at -20ºC in cryoprotection solution
(30% ethylene glycol, 30% glycerol, 30%dH2O, 10% PBS10X).

Immunofluorescence of BrdU

1. Wash 4x5 min with PBS1X

2. While the washes are being carried out, add 1ml of buffer * in a 2ml Eppendorf
and placed in the thermoblock until it reaches 80ºC→ (*Sodium Citrate 10mM,
0,05% TWEEN 20 PH6)

3. Place a slice per Eppendorf and incubate 30 min 80ºC 400rpm in the
thermoblock.

4. Once the incubation is over, let the slices warm to room temperature (do not
accelerate this process with ice) and remember to set the temperature of the
thermoblock to 37ºC.

5. Once the slices are tempered, transfer them to a 2ml tube with 1ml of 2N HCl
and incubate for 30 min at 37ºC 400rpm in the thermoblock.

6. Wash 6x5 min with PBS1X

7. Wash 6x5 min with PBS-T 0,3% (PBS with Triton X-100 #T8787)

8. Prepare the appropriate amount of block solution that we will use throughout the
immuno. (Newborn Calf Serum 5% diluted in PBS-T 0,3%) We will use 400uL
for the first block, 250uL for the primary antibody and 250uL for the secondary
antibody, all of this for one well.

9. Incubate 2h in blocking solution (5% NCS diluted in PBS-T 0,3%)

10. Incubate overnight at room temperature with the primary antibody dissolved in
blocking solution in a 1/500 ratio. (Anti-BrdU Rat monoclonal # AB6326 abcam
and Iba 1 rabbit # 5100342 Wako)

11. Wash 6x5 min with PBS-T 0,3%

12. Incubate 2 hours at room temperature with the secondary antibody dissolved in
blocking solution in a 1/500 ratio (Anti-rat Alexa 595 Donkey # A21209
Invitrogen and Anti-rabbit Alexa 488 goat # A11034 Invitrogen)

13. Wash 3x5 min with 0.3% PBS-T

14. Wash 3x5 min with PBS1X

15. Incubate 10 min with Dapi (at 1/10000 concentration in PBS)

16. Wash 3x5 min with PBS1X

17. Mount the immuno.