The muscles were frozen in 2‐methylbutan directly after removal. All cross‐sections were prepared on a cryostat at a thickness of 10 μm and stored at −20°C. For fibre‐type staining, the sections were first rehydrated with phosphate‐buffered saline (PBS) and then blocked for 30 min with blocking solution [0.4% Triton X‐100, 10% goat serum (Biological industries, Beit Haemek, Israel) in PBS] followed by a washing step with PBS. The primary antibodies against MyHC 2b (1:100; #BF‐F3; DSHB, Iowa, USA), MyHC 2a (1:200; #SC‐71; DSHB), MyHC 1 (1:50; BA‐D5; DSHB), and laminin (1:160; #ab11575; Abcam) were diluted in PBS containing 10% goat serum (Biological industries) and added for 1 h at RT. After a washing step with PBS, the secondary antibodies were added containing AF488 Gt anti‐Ms IgM (1:100; Invitrogen, Carlsbad, CA, USA), AF568 Gt anti‐Ms IgG1 (1:100; Invitrogen), DL405 Gt anti‐Ms IgG2b (1:50; Jackson), and AF647 Donkey anti‐Rabbit IgG (1:200; Jackson) diluted in PBS containing 10% goat serum. The slides were again washed with PBS and then mounted with Vectashield Hardset (H‐1600; Vector Labs, Burlingame, CA, USA). The entire section was imaged on a Zeiss (Oberkochen, Germany) Axio Scan.Z1 Slide Scanner.
Goat serum
Manufactured by Sartorius
Sourced in Israel
Goat serum is a biological reagent derived from the blood of goats. It contains a variety of proteins, growth factors, and other biomolecules that can be useful for cell culture and other research applications.
Automatically generated - may contain errors
Lab products found in correlation
Triton x, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Sartorius (2 mentions)
Ab7260, by Abcam (2 mentions)
Goat anti rabbit igg secondary antibody, by Proteintech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Proteintech (1 mentions)
Ix73 fluorescence microscope, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Biosharp (1 mentions)
Triton x 100, by Dingguo (1 mentions)
Ab66028, by Abcam (1 mentions)
Ab178847, by Abcam (1 mentions)
Cm1850, by Leica (1 mentions)
Ab9774, by Abcam (1 mentions)
Ab90810, by Abcam (1 mentions)
Ab11575, by Abcam (1 mentions)
Goat serum, by Jackson ImmunoResearch (1 mentions)
Af647 donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
5 protocols using goat serum
1
Muscle Fiber Characterization by Immunostaining
2
Isolation and Characterization of Cancer-Associated Fibroblasts
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LLC CAFs were obtained from the s.c. implanted LLC tumor by collagenase treatment, following separation with meshing by a nylon filter and adhesion of plastics, as previously described (1 (link),2 (link)). CAFs were incubated in DMEM media supplemented with 20% FBS at 37°C and passaged every 3–4 days. After 5 passages, cells were fixed with 4% paraformaldehyde (BioSharp Life Sciences) for 15 min, and subsequently permeabilized with 0.2% Triton-X-100 (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) for 15 min at room temperature. Cell were blocked with 10% goat serum (Biological Industries) at room temperature for 1 h and incubated with the following primary antibodies: Rabbit anti-α-smooth muscle actin (1:500; cat. no. NBP1-30894; Novus Biological, Ltd.), anti-FAP-α (1:500; cat. no. MAB9727; Novus Biological, Ltd.), anti-vimentin (1:500; cat. no. NBP1-97670; Novus Biological) or anti-pan cytokeratin (1:200; cat. no. NB600-579; Novus Biological) overnight at 4°C. Cells were washed three times with PBS and subsequently incubated with a goat anti-rabbit IgG secondary antibody (1:500; cat no. SA00001-2; ProteinTech Group, Inc.) for 1 h at 37°C. The nuclei of the CAFs were stained with DAPI (ProteinTech Group, Inc.) and cell images were captured using an OLYMPUS IX73 fluorescence microscope (magnification × 400; Olympus Corporation).
3
Histological Analysis of Brain Samples
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Brains were snap frozen in 2-methylbutane cooled in liquid nitrogen and embedded in OCT before cutting. Coronal sections (10 μm) were cut using a freezing sliding microtome (Leica CM1850) and stored at –20°C until use. For S100β and IBA1 immunostaining, slides were washed two times with PBS, blocked and permeabilized with PBS 1% bovine serum albumin, 5% goat serum (Biological Industries, Israel) and 0.05% Triton-X (Sigma-Aldrich) for 1 h and incubated with S100β antibody (1:500, ab66028, Abcam) or IBA1 antibody (1:200, ab178847, Abcam) overnight in 4°C. Slides were washed 3 times with PBS and incubated with secondary goat anti mouse antibody (1/700, Alexa-Flour) for 1 h at room temperature. DNA was stained with DAPI (1:1000, Sigma-Aldrich). For Thioflavin S (ThioS) staining, slides were incubated for 8 min with 0.01% ThioS solution in 50% ethanol. Slices were then briefly incubated twice for 10 s with 80% ethanol, and washed twice with double distilled water (DDW).
4
Immunofluorescence Staining of Brain Tissue
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Slides were thawed and washed twice with PBS; blocked and permeabilized with PBS 1% bovine serum albumin, 5% goat serum (Biological Industries, Beit Ha’emek, Israel) and 0.05% Triton-X (Sigma-Aldrich) for one hour; and incubated with the following primary antibodies overnight at 4 °C: αSMA (1/400, 19245, Cell Signaling, Danvers, MA, USA), GFAP (1/1000, ab7260, Abcam), MPO (1/100, ab90810, Abcam), RECA (1/100, ab9774, Abcam). Slides were washed three times with PBS and incubated with a fluorescent-labeled secondary antibody (1/700, Alexa-Flour) for 1 h at RT. For IgG staining, brains were incubated overnight with 1/200 Alexa-Flour Cy3 anti-rat IgG antibody. DNA was stained for 10 min with DAPI (1:1000, Sigma-Aldrich). Slides were mounted with Flouromount-G. αSMA and RECA images were used to calculate vessel area. In order to minimize the background signal, slides were incubated with secondary antibodies and without primary antibodies. This ensures that staining is produced from the detection of the antigen by the primary antibody and not by the detection system or the specimen.
5
Immunofluorescence Staining of GFAP
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Slides were thawed, washed twice with PBS, blocked and permeabilized with PBS 1% BSA, 5% goat serum (Biological Industries, Galilee, Israel) and 0.05% Triton-X (Sigma-Aldrich) for one hour, and incubated with GFAP (1/1000, ab7260, Abcam, Cambridge, UK) primary antibody overnight at 4 °C. The slides were subsequently washed 3 times with PBS and incubated with a fluorescent-labelled secondary antibody (1/700, Alexa-Flour) for 1 h at room temperature. DNA was stained for 10 min with DAPI (1:1000, Sigma-Aldrich), and the slides were mounted using Flouromount-G.
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