Peroxidase conjugated goat anti mice igg

Hypothalamus were homogenized in RIPA buffer and cleared by centrifugation, according to the standard techniques [28 (link)]. Western blots of whole tissue lysates were probed with primary antibodies against Kisspeptin1 (KISS1, Solarbio, K009431P), Kisspeptin1 receptor (KISS1R, Solarbio, K003544P), GnRH(PA5-97047, Thermo Fisher), GnRH receptor (abcam, ab183079), Tyrosine hydroxylase (TH, AB152; Merck Millipore), Arginine vasopressin (AVP, AB1565, Merck Millipore), Neuronal Nuclei (NeuN; MAB377, Merck Millipore), brain-derived neurotrophic factor (BDNF; AB203573, Abcam) and β-Tubulin (A01030HRP, Abbkine). The secondary antibody used was either peroxidase- conjugated goat anti-rabbit IgG (111-035-003; Jackson), or peroxidase-conjugated goat anti-mice IgG (115-035-003; Jackson). Protein markers (20351ES76; Shanghai Yisheng, China) were added on both sides of each gel to verify bands. The PVDF membranes were detected by enhanced che- moluminescence (Beyotime, China). Bands were analyzed using Image Lab™ Software (Bio-Rab Laboratories), were normalized to β-Tubulin and expressed as relative units (RU).

The total protein from hypothalamus and colon were separated by SDS-PAGE and then transferred to PVDF membrane. The membrane was soaked with skimmed milk powder to reduce the binding of non-specific antibodies. The membranes were then exposed to primary antibodies and secondary antibodies (peroxidase-conjugated goat anti-rabbit IgG (111–035-003, Jackson) or peroxidase-conjugated goat anti-mice IgG (115–035-003, Jackson) depending on the primary antibody). Protein was quantified with Lab image Software (BioRad, USA) and expressed as relative units to housekeeping proteins.
The primary antibodies are as follows: anti-free fatty acid receptor 2 (FFAR2; ABC299, Merck Millipore), anti-monocarboxylic acid transporter 1 (MCT1, ab93048, Abcam), anti-small peptide transporters (PEPT1, ab203043, Abcam), anti-GAPDH (A01020, Abbkine), and anti-β-tubulin (A01030HRP, Abbkine).

Hypothalamus was homogenized in Radio Immunoprecipitation Assay Lysis buffer (RIPA) buffer and cleared by centrifugation, according to the standard techniques. Western blots of whole tissue lysates were probed with primary antibodies against Kisspeptin (ab226786, Abcam, Cambridge, United Kingdom), GnRH (PA5-97047, Thermo Fisher Scientific, Waltham, MA, United States), β-Tubulin (A01030, Abbkine, CA, United States), GAPDH (A01020; Abbkine, CA, United States). The secondary antibody used was either peroxidase-conjugated goat anti-rabbit IgG (111-035-003; Jackson, West Grove, PA, United States), or peroxidase-conjugated goat anti-mice IgG (115-035-003; Jackson, West Grove, PA, United States). Protein markers (20351ES76; Shanghai Yisheng, China) were added on both sides of each gel to verify bands. The polyvinylidene fluoride (PVDF) membranes were detected by enhanced chemoluminescence (Beyotime, China). The bands were analyzed using Image LabTM Software (Bio-Rab Laboratories, Hercules, CA, United States), were normalized to β-Tubulin or GAPDH, and were expressed as relative units (RU).