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.

Hasil (Bahasa Indonesia) 2:

Imunohistokimia
Untuk imunohistokimia fluoresensi, bagian jaringan tipis
(5μm) dari karang gorgonian sehat deparaffinized dan
terhidrasi melalui serangkaian turun dari ethanols (dijelaskan
sebelumnya). Bagian yang permeabilized di 0,05% Triton
X-100 (Sigma Aldrich, St. Louis, MO) selama 15 menit pada suhu kamar.
Kemudian, bagian jaringan dibilas dalam air laut disaring
dan diinkubasi selama 1 jam pada suhu kamar dalam blocking
solusi (0,05 % Triton X-100, 3% dari serum kambing normal,
Jackson ImmunoResearch, West Grove, PA) dalam air laut disaring
untuk mencegah non-spesifik mengikat. Setelah memblokir, jaringan
bagian dibilas dengan air laut disaring selama 5 menit dan
kemudian diinkubasi dengan kelinci poliklonal antibodi primer
(1/500 diencerkan dalam menghalangi solusi) terhadap Aspergillus (Abcam,
Cambridge, MA) pada suhu 4oC selama 24 jam. Berikutnya, bagian jaringan yang
dibilas dengan air laut disaring selama 5 menit dan segera setelah itu, untuk mencegah fluoresensi endogen, bagian jaringan yang
diblokir dengan 0,1% sodium borohydrate selama 30 menit. Tissue
bagian dibilas lagi dengan air laut disaring selama 5 menit
dan diinkubasi selama 2 jam pada suhu kamar dengan Alexa
Fluor 546® (Invitrogen, Eugene, OR) kambing anti-kelinci sekunder
antibodi (1/200 diencerkan dalam menghalangi solusi) (Hidup Teknologi,
Carlsbad, CA). Akhirnya, bagian jaringan dicuci dua kali (30
s masing-masing) di disaring air laut dan kemudian diberi label dengan Alexa Fluor
488 Phalloidin (Hidup Teknologi, Carlsbad, CA) selama 5 menit, dibilas
lagi disaring air laut (2X setiap 30 s), dan inti yang
diwarnai dengan TO-PRO-3® (1/500 diencerkan dalam air laut disaring;
Hidup Teknologi, Carlsbad, CA). Slide kontrol disiapkan
tepat seperti yang dijelaskan tapi memanfaatkan antibodi primer
saja. Kontrol tambahan memanfaatkan antibodi sekunder saja,
juga dilakukan. Tidak ada pelabelan imunohistokimia yang
dilakukan pada A. cervicornis.