Alexa fluor 488 conjugated anti goat igg

The fixed brain tissues were sectioned as previously described in immunohistochemistry. Primary antibodies were diluted to 1:200 for anti-ionized calcium-binding adaptor molecule-1 (Iba1) (Wako Pure Chemicals, Osaka, Japan), for anti-neuronal nuclei (NeuN) (Merck Millipore Corporation, Darmstadt, Germany), and for anti-cleaved caspase-3 (Cell signaling, Danvers, MA, USA) antibodies. Brain tissues were soaked in diluted primary antibody solutions overnight at 4 °C and then washed with PBS three times. Sections were incubated with 1:300 of Alexa Fluor 488-conjugated anti-goat IgG (Thermo Fisher Scientific, Waltham, MA, USA) for anti-Iba1, 1:300 of rhodamine-labeled anti-mouse IgG (Merck Millipore Corporation, Darmstadt, Germany) for anti-NeuN, or 1:200 of Alexa Fluor 633-conjugated anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA) for anti-cleaved caspase-3 in PBS for 1 h at room temperature. The sections were then mounted on slides with a VECTASHIELD Antifade Mounting solution containing DAPI (Vector Laboratories, Burlingame, CA, USA) and examined under a Zeiss LSM 510 META microscope (Carl-Zeiss-Strasse, Oberkochen, Germany).

Free-floating sections of the spinal cord were blocked for 1 h at room temperature with 5% BSA in PBST to block non-specific binding, then incubated for 48 h at 4°C with a mixture of rabbit polyclonal antibody against rat BNP (diluted 1:2000; Millipore) and either guinea-pig polyclonal CGRP antibody (1:200; T-5053, Peninsula Lab, San Carlos, CA, USA) or goat anti-ChAT antibody (diluted 1:500; Millipore). After three rinses with PBST, sections were incubated for 3 h at room temperature with the appropriate secondary antibodies. The secondary antibodies were Alexa Fluor 555-conjugated anti-rabbit IgG, Alexa Fluor 488-conjugated anti-guinea pig IgG, or Alexa Fluor 488-conjugated anti-goat IgG (diluted 1:500, Thermo Fisher Scientific Inc., Waltham, MA, USA). Negative control sections were subjected to the same procedures without primary antibody. After three washes with PBST, the sections were mounted on gelatin-coated glass slides and examined using a confocal laser-scanning microscope (LSM 510 META, Carl Zeiss, Heidelberg, Germany).

Overnight S. pneumoniae (TIGR4 isogenic strains) cultures were used to inoculate liquid THY cultures. Cultures were grown to an OD₆₂₀ of 0.2 and then washed twice in 1× PBS. Approximately 10 × 10⁶ cells were collected for staining. For fH binding to whole bacteria, S. pneumoniae cells were incubated with IgG-depleted normal human serum (Quidel) for 1 h at 37°C. Following incubation, cells were washed twice in PBS containing 2% fetal bovine serum (FBS) and incubated with goat antisera against human factor H (Quidel) for 30 min at 37°C. Cells were then incubated with Alexa Fluor 488-conjugated anti-goat IgG (Thermo Fisher) (clone A11055). For binding of anti-PspC rabbit antibodies to whole bacteria, ~10 × 10⁶ S. pneumoniae cells were incubated with purified anti-PspC IgG for 30 min at 37°C. Afterward, cells were washed twice in PBS containing 2% FBS and stained with goat anti-rabbit IgG FITC-conjugated antibody (Abcam, Inc.) (clone ab6717). Cells were incubated at 4°C for 30 min and then washed with PBS containing 2% FBS. Finally, the cells were fixed in fixation buffer (data were immediately acquired using an LSR flow cytometer [BD Biosciences]). Data were analyzed with FlowJo software (Tree Star, San Carlos, CA).