The eyes for immunostaining were enucleated 7 or 14 days after photocoagulation and then fixed in 4% paraformaldehyde for 12 h. The RPE-choroid-sclera complex was separated from the retina, isolated, and blocked in non-immune horse serum (Vector Labs) for 1 h, and then incubated with the primary antibody at 4 °C overnight. Then, the RPE-choroid-sclera complex was stained with a secondary antibody for 1 h, and flat-mounted on the slide. The slides were viewed and photographed for an overall picture and FLUOVIEW FV10i (Olympus) for the laser spots. The intensity of CD68 and VEGF-A in the CNV lesion was determined by the ImageJ analysis software (National Institutes of Health). The accumulation of Iba-1+ myeloid cells around the CNV was counted in Iba-1-stained whole RPE-choroidal flat mounts viewed from the RPE side. The primary antibodies used were as follows: rabbit anti-Iba1 (1:200; FUJIFILM Wako, catalog 019-19741), sheep anti-mPGRN (1:200; R&D systems, catalog AF2557), rabbit anti-VEGF-A (1:200; Merck Millipore, catalog PC315), rat anti-CD68 (1:200; Bio-Rad, catalog MCA1957GA), Alexa Fluor® 647 donkey anti-sheep IgG (1:1000; Invitrogen, catalog A21448), Alexa Fluor® 647 donkey anti-rat IgG (1:1000; Jackson ImmunoResearch, catalog 712-605-153), and Alexa Fluor® 546 donkey anti-rabbit IgG (1:1000; Invitrogen, catalog A10040).
Non immune horse serum
Manufactured by Vector Laboratories
Non-immune horse serum is a laboratory reagent used as a base material for various biological applications. It serves as a source of proteins, nutrients, and other components essential for cell culture and other in vitro experiments.
Automatically generated - may contain errors
Lab products found in correlation
Fluoview fv10i, by Olympus (3 mentions)
Alexa fluor 546 donkey anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Bz x710, by Keyence (2 mentions)
Alexa fluor 647 donkey anti rat igg, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 647 donkey anti sheep igg, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Mas coat, by Matsunami (1 mentions)
3 protocols using non immune horse serum
1
Immunostaining of Choroidal Neovascularization
2
Immunostaining of Ocular Tissues
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For immunostaining the ocular tissues, the eyes were enucleated and fixed in 4% paraformaldehyde for at least 24 h at 4⁰ C, and then immersed in 25% sucrose in 0.01 M phosphate buffered sarin (PBS) for 2 days. The eyes were then embedded in optimal cutting temperature (OCT) compound (Sakura Finetek Japan) and immediately frozen with liquid nitrogen. Ten micrometer sections were cut with a cryostat, and the sections were mounted on glass slides (MAS COAT; Matsunami Glass).
The retinal sections were blocked in non-immune horse serum (Vector Labs) for 1 h, and then incubated with the primary antibody at 4⁰ C overnight. The next morning the sections were covered with a secondary antibody for 1 h and then counterstained with Hoechst 33342 (1:1000; Invitrogen, catalog H3570) for 15 min.
The following antibodies were used; sheep anti-mPGRN (1:100; R&D Systems, catalog AF2557), rabbit anti-Iba1 (1:200; FUJIFILM Wako Chemicals, catalog 019-19741), Alexa Fluor ® 647 donkey anti-sheep IgG (1:1000; Invitrogen, catalog A21448), and Alexa Fluor ® 546 donkey anti-rabbit IgG (1:1000; Invitrogen, catalog A10040). The immunostained sections were examined and photographed with a confocal microscope (FLUOVIEW FV10i; Olympus) or BZ-X710 (Keyence).
The retinal sections were blocked in non-immune horse serum (Vector Labs) for 1 h, and then incubated with the primary antibody at 4⁰ C overnight. The next morning the sections were covered with a secondary antibody for 1 h and then counterstained with Hoechst 33342 (1:1000; Invitrogen, catalog H3570) for 15 min.
The following antibodies were used; sheep anti-mPGRN (1:100; R&D Systems, catalog AF2557), rabbit anti-Iba1 (1:200; FUJIFILM Wako Chemicals, catalog 019-19741), Alexa Fluor ® 647 donkey anti-sheep IgG (1:1000; Invitrogen, catalog A21448), and Alexa Fluor ® 546 donkey anti-rabbit IgG (1:1000; Invitrogen, catalog A10040). The immunostained sections were examined and photographed with a confocal microscope (FLUOVIEW FV10i; Olympus) or BZ-X710 (Keyence).
3
Immunostaining of RPE-choroid-sclera complex
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The eyes for immunostaining were enucleated 7 or 14 days after photocoagulation and then fixed in 4% paraformaldehyde for 12 h. The RPE-choroid-sclera complex was separated from the retina, isolated, and blocked in non-immune horse serum (Vector Labs) for 1 h, and then incubated with the primary antibody at 4⁰ C overnight.
Then the RPE-choroid-sclera complex was stained with a secondary antibody for 1 h, and flat-mounted on the slide. The slides were viewed and photographed with the BZ-X710 (Keyence) and FLUOVIEW FV10i (Olympus) microscopes. The intensity of CD68 and VEGF-A in the CNV lesion was determined by the ImageJ analysis software (National Institutes of Health). The accumulation of Iba-1 + myeloid cells around the CNV were counted in Iba-1 stained whole RPE-choroidal flat mounts viewed from the RPE side. The primary antibodies used were: rabbit anti-Iba1
Then the RPE-choroid-sclera complex was stained with a secondary antibody for 1 h, and flat-mounted on the slide. The slides were viewed and photographed with the BZ-X710 (Keyence) and FLUOVIEW FV10i (Olympus) microscopes. The intensity of CD68 and VEGF-A in the CNV lesion was determined by the ImageJ analysis software (National Institutes of Health). The accumulation of Iba-1 + myeloid cells around the CNV were counted in Iba-1 stained whole RPE-choroidal flat mounts viewed from the RPE side. The primary antibodies used were: rabbit anti-Iba1
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