ImmunohistochemistryFor fluorescenc

Immunohistochemistry
For fluorescence immunohistochemistry, thin tissue sections
(5μm) from healthy gorgonian coral were deparaffinized and
hydrated through a descending series of ethanols (explained
previously). Sections were permeabilized in 0.05% Triton
X-100 (Sigma Aldrich, St. Louis, MO) for 15 min at room temperature.
Then, tissue sections were rinsed in filtered seawater
and incubated for 1 hour at room temperature in a blocking
solution (0.05% Triton X-100; 3% of normal goat serum;
Jackson ImmunoResearch, West Grove, PA) in filtered seawater
to prevent non-specific binding. After blocking, the tissue
sections were rinsed with filtered seawater for 5 min and
then incubated with a rabbit polyclonal primary antibody
(1/500 diluted in blocking solution) against Aspergillus (Abcam,
Cambridge, MA) at 4oC for 24 hours. Next, tissue sections were
rinsed with filtered seawater for 5 min and immediately after,to prevent any endogenous fluorescence, tissue sections were
blocked with 0.1% sodium borohydrate for 30 mins. Tissue
sections were rinsed again with filtered seawater for 5 min
and incubated for 2 hours at room temperature with Alexa
Fluor 546® (Invitrogen, Eugene, OR) goat anti-rabbit secondary
antibody (1/200 diluted in blocking solution) (Life Technology,
Carlsbad, CA). Finally, tissue sections were washed twice (30
s each) in filtered seawater and then labeled with Alexa Fluor
488 Phalloidin (Life Technology, Carlsbad, CA) for 5 min, rinsed
again in filtered seawater (2X each 30 s), and nuclei were
stained with TO-PRO-3® (1/500 diluted in filtered seawater;
Life Technology, Carlsbad, CA). Control slides were prepared
precisely as described but utilizing the primary antibody
only. Additional controls utilizing secondary antibodies only,
were also performed. No immunohistochemical labeling was
performed on A. cervicornis.

Hasil (Bahasa Indonesia) 1:

ImmunohistochemistryFor fluorescence immunohistochemistry, thin tissue sections(5μm) from healthy gorgonian coral were deparaffinized andhydrated through a descending series of ethanols (explainedpreviously). Sections were permeabilized in 0.05% TritonX-100 (Sigma Aldrich, St. Louis, MO) for 15 min at room temperature.Then, tissue sections were rinsed in filtered seawaterand incubated for 1 hour at room temperature in a blockingsolution (0.05% Triton X-100; 3% of normal goat serum;Jackson ImmunoResearch, West Grove, PA) in filtered seawaterto prevent non-specific binding. After blocking, the tissuesections were rinsed with filtered seawater for 5 min andthen incubated with a rabbit polyclonal primary antibody(1/500 diluted in blocking solution) against Aspergillus (Abcam,Cambridge, MA) at 4oC for 24 hours. Next, tissue sections wererinsed with filtered seawater for 5 min and immediately after,to prevent any endogenous fluorescence, tissue sections wereblocked with 0.1% sodium borohydrate for 30 mins. Tissuesections were rinsed again with filtered seawater for 5 minand incubated for 2 hours at room temperature with AlexaFluor 546® (Invitrogen, Eugene, OR) goat anti-rabbit secondaryantibody (1/200 diluted in blocking solution) (Life Technology,Carlsbad, CA). Finally, tissue sections were washed twice (30s each) in filtered seawater and then labeled with Alexa Fluor488 Phalloidin (Life Technology, Carlsbad, CA) for 5 min, rinsedlagi di disaring air laut (2 X 30 setiap s), dan intidiwarnai dengan TO-PRO-3 ® (1/500 diencerkan dalam disaring air;Hidup teknologi, Carlsbad, CA). Kontrol slide disusuntepat seperti yang dijelaskan tapi memanfaatkan antibodi utamahanya. Kontrol tambahan yang memanfaatkan sekunder antibodi saja,juga dilakukan. Tidak ada label imunohistokimia adalahdilakukan pada A. cervicornis.