For immunohistochemistry, explants or free-floating tissue sections were rinsed in PBS. For myelin proteins, tissues were permeabilized 10% Triton X-100 in PBS (PBSTx) for 20 min and then rinsed in PBS. Tissues are blocked with 5% normal donkey serum (NDS) in PBSTx (1%) for 1h, and then incubated with primary antibodies overnight at room temperature. Following 3, 10 min washes in PBS, Alexa Fluor-labeled secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were applied (1:800) 2h at room temperature, washed 3 times for 10 min in PBS, mounted on slides with water, dried and cover-slipped with Fluoromount G (Southern Biotech, Birmingham, AL). The following primary antibodies were used: Rat anti-PLP 1:100 (Yamamura et al., 1991 (link)), Rabbit anti-S100b 1:500 (Sigma, S2644), Rabbit anti-GFAP 1:1000 (Sigma, G9269), Goat anti-Iba1 1:400 (Abcam, ab5076), Guinea Pig anti-NG2 1:2000, Rabbit anti-NG2 1:2000, and Rabbit anti-PDGFRa 1:500 (Gifts from William Stallcup, Burnham Institute).
Alexa fluor labeled secondary antibodies
Manufactured by Jackson ImmunoResearch
Sourced in Panama, United States
Alexa Fluor-labeled secondary antibodies are fluorescent-conjugated antibodies designed to detect and visualize primary antibodies in various immunoassays. These secondary antibodies are labeled with Alexa Fluor dyes, which provide bright and photostable fluorescence signals for sensitive detection.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti gfap, by Merck Group (2 mentions)
Rabbit anti s100b, by Merck Group (2 mentions)
Goat anti iba1, by Abcam (2 mentions)
Fluoromount g, by Southern Biotech (2 mentions)
Goat anti sox10, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti mog, by Abcam (1 mentions)
Rabbit anti gst pi, by Enzo Life Sciences (1 mentions)
Mouse anti aqp4, by Santa Cruz Biotechnology (1 mentions)
Mouse anti mbp, by Fortrea (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Vectashield hard set containing dapi, by Vector Laboratories (1 mentions)
4 protocols using alexa fluor labeled secondary antibodies
1
Immunohistochemical Labeling of Glial Cells
2
Cerebellar Slice Immunohistochemistry Protocol
3
Immunofluorescence Staining of Murine Tracheal Epithelial Cells
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MTEC on inserts were rinsed with PBS followed by fixation with 4% (w/v) paraformaldehyde (PFA) for 10 min at room temperature. After fixation cells were washed 3 times with PBS/0.2% (v/v) Triton-X100 (Sigma-Aldrich) to remove residual mucus from the apical surface. Non-specific binding sites were blocked with 5% (v/v) donkey serum (EMD Millipore, Billerica, MA, USA), 1% (w/v) BSA and 0.2% (v/v) Triton-X100 in PBS for 30 min at room temperature, followed by incubation with primary antibodies in blocking buffer at 4 °C overnight (Supplemental Table 2 ). The next day, MTEC were washed three times with PBS/0.02% Triton X-100 and incubated for 2 h at room temperature with Alexa Fluor labeled secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) in blocking buffer (Supplemental Table 2 ). After three washing steps with PBS/0.02% (v/v) Triton-X100 and one washing step with PBS, the cells were mounted with Vectashield hardset containing DAPI (Vector Laboratories, Burlingame, CA, USA). Images were acquired using a Leica SP5 confocal microscope. Cell counting was performed using ImageJ and 10–15 areas from each insert were analyzed for each replicate.
4
Comprehensive Tissue Staining Protocols
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For hematoxylin and eosin (H&E) staining, the paraffin sections were de-waxed using graded xylene solutions. Staining was performed according to the manufacturer’s instructions (Solarbio Science and Technology, Beijing, China).
For immunohistochemistry, paraffin sections were prepared by the above procedures, microwaved in sodium citrate buffer, and incubated with primary and secondary antibodies (Boster Biological Technology, Wuhan, China). Finally, the sections were developed using the AEC (3-amino-9-ethylcarbazole) Staining Kit (Boster Biological Technology, Wuhan, China).
For immunofluorescence, cryosections were thawed at room temperature (26 °C), rehydrated in 0.1 mol·L−1 phosphate-buffered saline and microwaved in sodium citrate buffer. After incubation with the primary antibody, the sections were incubated with Alexa Fluor-labeled secondary antibodies (Jackson Laboratory, Pennsylvania, USA).
For immunohistochemistry, paraffin sections were prepared by the above procedures, microwaved in sodium citrate buffer, and incubated with primary and secondary antibodies (Boster Biological Technology, Wuhan, China). Finally, the sections were developed using the AEC (3-amino-9-ethylcarbazole) Staining Kit (Boster Biological Technology, Wuhan, China).
For immunofluorescence, cryosections were thawed at room temperature (26 °C), rehydrated in 0.1 mol·L−1 phosphate-buffered saline and microwaved in sodium citrate buffer. After incubation with the primary antibody, the sections were incubated with Alexa Fluor-labeled secondary antibodies (Jackson Laboratory, Pennsylvania, USA).
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