Rabbit anti iba1

Immunodetection of S-opsin⁺OS using S-opsin antibody (goat anti-OPN1SW; 1:1000; Santa Cruz Biotechnologies, Heidelberg, Germany) and reactive-Iba-1⁺ monocytic cells using Iba-1 antibody (1:500; rabbit anti-Iba-1, Abcam, Cambridge, UK) detected with Alexa Fluor-594 donkey anti-goat and Alexa Fluor-488 donkey anti-rabbit (1:500; IgG (H + L), Molecular Probes Invitrogen, Barcelona, Spain), respectively, was performed on whole mounts following previously described protocols [15 (link),18 (link),25 (link)].

After the behavioural tests, the mice were anaesthetized and transcardially perfused with saline. Then, the brains were removed, and the hemispheres were separated. Brain samples assigned for immunohistochemical staining were immersed in OCT at -80 °C before coronal sectioning on a Frozen slicer (Thermo Fisher Scientific, USA) (10 μm thickness). The sections were restored to room temperature, fixed with cold acetone for 5 min, and permeabilizing agent was used for 5 min. Then, the cells were rinsed three times at room temperature for 5 min each with 1x PBS (pH 7.2-7.4) and blocked with 10% bovine serum in 0.1% Triton X-100 for 30 min. The blocking solution covered all brain tissues and prevented the tissue from drying out. Then, the samples were incubated with primary antibodies overnight at 4 °C. The primary antibodies included rabbit anti-NeuN (1:500, Abcam), rabbit anti-GFAP (1:200, CST), rabbit anti-Iba-1 (1:100, Abcam), rabbit anti-CD68 (1:300, Abcam), rabbit anti-APP (1:100, Millipore), and mouse anti-β-amyloid specific for Aβ₄₂ (1:200, CST). The next morning, the sections were incubated for 1 h at room temperature with DyLight 488-/555-conjugated goat anti-rabbit/mouse IgG (1:200, Abcam) and stained with DAPI for 3 min. Fluorescence images were captured using an Olympus FV3000 confocal microscope.

Double immunofluorescence analysis was performed to detect HMGB1 and a neuronal marker (NeuN), a microglial marker (Iba1), or an astrocytic marker (GFAP) as previously described [2 (link)]. Briefly, 30-μm-thick sections were incubated overnight at 4 °C with the primary mouse anti-HMGB1 (MyBioSource, CA, USA) and rabbit anti-NeuN, rabbit anti-Iba1, or rabbit anti-GFAP (Abcam, Cambridge, MA, USA) antibodies. Then, the sections were incubated with Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 594-conjugated donkey anti-mouse IgG (Molecular Probes, MA, USA) for 2 h at room temperature. Additionally, the interaction between HMGB1 and TLR3 was explored using primary rabbit anti-HMGB1 and mouse anti-TLR3 antibodies as described above. Images were captured using a Leica confocal microscope (Leica Microsystems, Buffalo Grove, IL, USA).

The dewaxing, rehydration and antigen retrieval procedure was carried out as above. Sections were labeled with the appropriate combination of 1:200 mouse anti-MBP (Cat# MAB381, Millipore), 1:200 rabbit anti-Iba1 (Cat# ab178846, Abcam, Cambridge, England), or rabbit anti-GFAP (Cat# ab68428, Abcam), incubated overnight at 4 °C. The appropriate secondary antibodies were added; Alexa Fluor 488 goat anti-rabbit (Cat# A11008, Invitrogen, Carlsbad, California, USA), Alexa Fluor 568 goat anti-mouse (Cat# A11004, Invitrogen) and/or Alexa Fluor 647 goat anti-rabbit (Cat# A32733 Invitrogen) (1:200) incubated for 3 h at room temperature. Hoechst (Sigma) was applied to sections for 10 min, then mounted with Vectashield (Vector Laboratories).

We visualized NLRP3 inflammasomes in microglia, neurons, and astrocytes through triple immune-fluorescence staining of brain tissue sections (n=6/group) for NLRP3, an apoptosis-associated speck-like protein containing a CARD (ASC), and IBA-1, NeuN, or GFAP. Goat anti-NLRP3 (1:500, Millipore), mouse anti-ASC (1:1000, Santa Cruz), rabbit anti-NeuN (1:1000, Millipore), rabbit anti-GFAP (1:1000, Millipore), rabbit anti-iba1 (1:1000 Abcam), donkey anti-goat IgG tagged Alexa Fluor 488 (1:200, Invitrogen), donkey anti-mouse Alexa Fluor 594 (1:200, Invitrogen), and donkey anti-rabbit 405 (1:200, Invitrogen) were employed in these studies. Two-micrometer thick optical Z-sections were employed for blinded quantification of the total number of NLRP3 inflammasomes (i.e., structures positive for both NLRP3 and ASC) in 216 μm² area of the CA3 subfield of the hippocampus (2 sections/animal, n=5-6/group). We also quantified the percentages of IBA-1+ microglia, NeuN+ neurons, and GFAP+ astrocytes containing NLRP3 inflammasomes (2 sections/animal) [25 (link)].

Cells were plated on a black glass bottom 96-well plate at a density of ~ 30,000 cells/cm². On Day 8, cells were washed with 1× phosphate buffered solution (PBS) and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Permeabilization of the cells was done with 1× PBS containing 0.25% Triton™X-100. Following 1 h blocking in 1× PBS with 5% goat serum and donkey serum at room temperature, primary rabbit anti-IBA1 (Abcam plc, Cat. No. ab178846; 1:1000), rabbit anti-TREM2 (Cell Signaling, Cat. No. D8I4C; 1:200), and rabbit anti-P2Y12 (Thermo Fisher Scientific Inc, Cat. No. 4H5L19; 1:100) antibodies were added in the blocking solution and incubated at 4 °C overnight. The next day, cells were washed 3 times with PBS for 5 min and stained with donkey anti-rabbit Alexa Fluor^® conjugated secondary antibody (Thermo Fisher Scientific Inc., Cat. No. A-21206) at 1:500 for 1 h at room temperature in the dark. After secondary antibody staining, cells were washed 3 times with PBS and imaged on a Zeiss^® LSM 710 microscope. Images were segmented and quantified using CellProfiler™.